KR102410132B1 - Hyraluronic acid dermal filler composition for tissue restoration - Google Patents

Abstract

The present invention relates to a skin filler composition for tissue repair, and the skin filler containing the polynucleotide and hyaluronic acid of the present invention has superior viscosity and elasticity than the hyaluronic acid skin filler, and It was confirmed that the synthesis ability was excellent, and the compression phenomenon due to the initial swelling during the procedure was reduced.

Description

Hyaluronic acid skin filler composition for tissue repair

The present invention relates to a hyaluronic acid dermal filler composition for tissue repair.

The soft tissue of the human body maintains its structure by the extracellular matrix including proteins such as collagen and elastin and glycosaminoglycans. When a defect occurs, the shape can be restored and corrected by expanding the soft tissue by inserting a living tissue or synthetic polymer chemical into the relevant site. In this regard, a substance used for wrinkle improvement or contour correction by injecting a component similar to skin tissue into a specific site and expanding soft tissue is generally called a dermal filler or filler. can be classified into the following two types according to the mechanism of action showing the effect. One is a skin filler that produces an enlargement effect by directly increasing the volume of the injected substance. There are skin fillers.

In addition, fillers are also used for cosmetic purposes according to skin aging. Although surgery or botox injections are used for similar purposes, the surgery has the disadvantages of leaving scars and taking a long time, and botox has many side effects because it uses a neurotoxin. On the other hand, in the case of a filler using a polymer existing in a living body, it is widely used because it has fewer side effects and can save time with a simple procedure.

The first filler was autologous fat grafting in the 1890s, and then paraffin and liquid silicone were used. After that, fillers using collagen typified by Zyderm and Zyplast were invented, but since collagen fillers use animal-derived collagen, an allergy test must be performed one month before the procedure, and the lifespan is relatively short, about 3 to 6 months (Baumann, L., J. Kaufman, and S. Saghari, Collagen fillers. Dermatologic therapy, 2006. 19(3): p. 134-140.). To compensate for this, a hyaluronic acid-based filler was developed. Representative examples include Hylaform and Restylane/Perlane, which have a lifespan of 6 to 12 months compared to collagen, which is twice as long, and has the advantage of not having an allergy test.

On the other hand, the present inventors were studying a method to improve the efficacy of the previously used hyaluronic acid skin filler, and when mixing hyaluronic acid and (polynucleotide), the physical properties and injection power are superior to that of the filler of hyaluronic acid alone, The present invention was completed by confirming that the collagen synthesis ability is excellent when injected into a mouse living body, and there is little initial swelling after injection.

It is an object of the present invention to provide a hyaluronic acid dermal filler composition for tissue repair.

In order to achieve the above object, the present invention provides a dermal filler composition for tissue repair containing (polynucleotide) and hyaluronic acid as active ingredients.

The present invention relates to a skin filler composition for tissue repair, and the filler containing hyaluronic acid and (polynucleotide) of the present invention has superior viscosity and elasticity than the filler of hyaluronic acid alone, and collagen in cells and in vivo. It was confirmed that the synthesizing ability of the was significantly increased, and the compression phenomenon caused by the initial swelling during the procedure was reduced.

1 is a diagram confirming the difference between G* (Complex modulus), G' (Storage modulus), G'' (Loss modulus) and δ (Phase angle) among rheological properties between samples.
Figure 2 is a diagram confirming the viscoelasticity (Viscoelasticity) of the rheological properties of the sample according to the frequency sweep:
JUVEDERM VOLUMA®: Positive control (hyaluronic acid 20 mg/ml)
JUVEDERM VOLBELLA®: positive control (hyaluronic acid 15 mg/ml)
HA-PN 0.1%: hyaluronic acid (HA) 1.5% and DNA polynucleotide (PN) 0.1% mixed sample;
HA-PN 0.5%: mixed sample of HA 1.5% and PN 0.5%;
HA-PN 1%: mixed sample of HA 1.5% and PN 1%;
HA 5%-PN 5%: mixed sample of HA 5% and PN 5%;
HA 10%-PN 0.1%: mixed sample of HA 10% and PN 0.1%;
HA-PDRN 0.1%: HA 1.5% and PDRN (Polydeoxyribonucleotide) 0.1% mixed sample;
HA-PDRN 0.5%: HA 1.5% and PDRN 0.5% mixed sample; and
HA-PDRN 1%: A mixed sample of 1.5% HA and 1% PDRN.
3 is a diagram showing the analysis of G', G'', ŋ* (complex viscosity) and δ among the rheological properties of the sample according to a frequency sweep:
G': Storage modulus, rheometer (Kinexus, Malvern, UK) with a stainless steel plate, gap of 0.5mm, frequency 0.1~10Hz;
G'': Loss modulus, rheometer (Kinexus, Malvern, UK) with a stainless steel plate, gap of 0.5mm, frequency 0.1~10Hz;
ŋ*: Complex viscosity, rheometer (Kinexus, Malvern, UK) with a stainless steel plate, gap of 0.5mm, frequency 0.1~10Hz; and
δ: Phase angle, rheometer (Kinexus, Malvern, UK) with a stainless steel plate, gap of 0.5mm, frequency 0.1~10Hz.
4 is a diagram confirming the difference between G*, G', G'' and δ among the rheological properties between the HA-PN sample and the HA-PDRN sample.
5 is a diagram illustrating the analysis of G', G'', ŋ* and δ among the rheological properties of the HA-PN sample and the HA-PDRN sample according to a frequency sweep.
Figure 6 is a diagram confirming the collagen synthesis ability by the treatment of the sample in human dermal fibroblasts (HDF):
BL: Blank (cell culture medium);
NT: negative control group (untreated group);
HA: the experimental group treated with hyaluronic acid 0.025%, 0.05% or 0.1% sample;
PN: Experimental group treated with DNA polynucleotide 0.1%, 0.5%, or 1% sample;
HA-PN: an experimental group treated with a mixed sample of 1.5% HA and 0.1% PN, a mixed sample of 1.5% HA and PN 0.5%, or a mixed sample of 1.5% HA and 1% PN; and
Vit100: Vitamin C 100 μM treatment group (positive control group)
7 is a diagram confirming the expression of collagen protein by treatment of a sample in HDF-N (Human Dermal Fibroblast, Neonnatal) cells:
HA: hyaluronic acid;
PN: DNA polynucleotide.
8 is a diagram showing an experimental schedule for the evaluation of persistence and tissue staining experiments in mice injected with the sample.
9 is a view showing an enlarged photograph of a PN sample injected into a mouse living body (Folliscope (LeedM, Seoul, Korea), x15).
10 is a view showing an enlarged photograph of a positive control group injected into a mouse in vivo (Folliscope (LeedM, Seoul, Korea), x15).
11 is a 3D photo simulation of a sample injected into a mouse living body (with Software PRIMOS 5.8).
12 is a diagram illustrating an initial volume change after injection of a sample into a mouse living body (Volumetric analysis; PrimosLITE Topography with Software PRIMOS 5.8).
13 is a diagram confirming the collagen-forming ability over time after injection of a PN sample into a mouse living body (MT stain; X100).
14 is a diagram confirming the collagen-forming ability according to time after injection of the HA 1.5%-PN 0.1% sample into the mouse living body (MT stain; X100, X200)
15 is a diagram confirming the collagen-forming ability according to time after injection of the HA 1.5%-PN 0.5% sample into the mouse living body (MT stain; X100, X200).
FIG. 16 is a diagram confirming the collagen-forming ability according to time after injection of the HA 1.5%-PN 1% sample into the mouse living body (MT stain; X100, X200).
17 is a diagram confirming the collagen-forming ability over time after injection of JUVEDERM VOLUMA® into a mouse (MT stain; X100, X200).
18 is a diagram confirming the collagen-forming ability over time after injection of JUVEDERM VOLLBELLA® into a mouse living body (MT stain; X100, X200).
19 is a diagram confirming the expression level of TRVP4 in a biopsy tissue 4 weeks after injection of a sample into a mouse living body (immunohistofluorescence staining; X 100).
20 is a diagram confirming the expression level of TRVP4 in the biopsy tissue 4 weeks after the injection of the sample into the mouse (immunohistofluorescence staining; X 200).
21 is an electrophoresis result confirming the size of the polynucleotide used in the present invention:
lane 1: PDRN;
Lane 2: PN of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a dermal filler composition for tissue repair containing a polynucleotide and hyaluronic acid as active ingredients.

As used herein, 'filler' means used for the purpose of increasing tissue volume (eg, tissue enlargement) and/or improving tissue function, and improving moldability. Therefore, the dermal filler composition for tissue repair of the present invention can be used to increase the tissue volume or improve the tissue function of soft tissues medically or cosmetically, and in particular, remove or improve skin wrinkles; It can be used to enlarge the volume of body parts such as cheeks, lips, chest, and buttocks.

In addition, in the present specification, the term 'hyaluronic acid' is used to include both hyaluronic acid itself as well as salts and derivatives thereof. Therefore, the term 'hyaluronic acid' used below is a concept including an aqueous solution of hyaluronic acid, an aqueous solution of a hyaluronic acid salt, and an aqueous mixture thereof. The hyaluronic acid salt may be at least one selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and tetrabutylammonium hyaluronate.

Although the molecular weight of the hyaluronic acid of the present invention is not particularly limited, a range of 500 to 6,000 kilodaltons may be used to implement various physical properties and biocompatibility, and more specifically, it may be 900 to 1,100 kilodaltons.

In addition, the hyaluronic acid may be included in an amount of 0.01 to 5.0% by weight of the total weight of the composition, specifically, it may be included in an amount of 0.05 to 2% by weight, and more specifically, it may be included in an amount of 1 to 2% by weight. When it exceeds 2% by weight, the viscous properties of the formulation rapidly decrease and the elastic properties increase, resulting in excessive tissue pressure and unnaturalness of the treatment site.

In addition, the polynucleotide may be included in 0.01 to 3% by weight of the total weight of the composition, specifically, may be included in 0.05 to 2% by weight, more specifically, may be included in 0.1 to 1% by weight, more Specifically, it may be included in an amount of 0.3 to 0.7% by weight. If it exceeds the corresponding range, viscous properties and elastic properties may decrease, thereby reducing the effect of improving skin wrinkles. The remainder of the composition may be composed of a pharmaceutically suitable carrier such as water or physiological saline.

"Polynucleotide" of the present invention refers to a polymer composed of nucleotide units as understood by one of ordinary skill in the art. Preferably, it may include nucleotide units including adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). In addition, the unit may be modified, and the modified nucleotide units are 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouri Dean, 5-carboxymethylamino-methyluridine, dihydrouridine, 2-O-methylpseudouridine, 2-O-methylguanosine, inosine, N6-isopentyladenosine, 1-methyladenosine, 1-methyl Pseudouridine, 1-methylguanosine, 1-methyl inosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine , 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-methoxyuridine, 5-methoxycarbonylmethyl-2-thiouridine, 5 -Methoxycarbonylmethyluridine, 2-methylthio-N6-isopentyladenosine, uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid, wybutoxosine, wybutosine ( wybutosine), pseudouridine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2-O -methyl-5-methyluridine, or 2-0-methyluridine, etc., and the like.

The polynucleotides of the present invention are naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and may be single-stranded or double-stranded. It may also include nucleic acid analogs, wherein the nucleic acid analogs are non-naturally occurring bases, nucleotides involved in linkage with nucleotides other than naturally occurring phosphodiester bonds, or phosphodiesters. Included are nucleic acid analogs comprising nucleotides comprising bases attached via linkage rather than bond. The nucleotide analogues are, for example, phosphorothioate, phosphorodithioate, phosphorotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral -methyl phosphonate, 2-O-methyl ribonucleotide, or peptide-nucleic acid (PNA), and the like. The polynucleotides of the invention may also be polynucleotides comprising synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art.

The molecular weight of the polynucleotide of the present invention may be 350 to 2,300 kilodaltons, more specifically It may be 250 to 2100 kilodaltons, more specifically 480 to 2,000 kilodaltons. If it is less than 480 kilodaltons, the efficiency of improving the viscoelasticity of the formulation is lowered, and if it exceeds 2,000 kilodaltons, the injection pressure may increase due to excessive viscosity increase.

Since the filler composition comprising hyaluronic acid and polynucleotide of the present invention has excellent long-term durability as well as biocompatibility, a certain form without any side effects when introduced into the body, such as a wrinkle improving agent, a cosmetic agent, a joint function improving agent, and an anti-adhesion agent It can be used as a medical, cosmetic, or cosmetic material to maintain, and preferably can be usefully used as a filler material that can be injected into the skin soft tissue.

In a specific embodiment of the present invention, the HA and PN mixed samples have all the G' (Storage modulus), G'' (Loss modulus), G* (Complex modulus) and δ (Phase angle) values than the control hyaluronic acid filler. It was measured to be high (see Table 3 and FIGS. 1 to 5), and it was confirmed that the HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5% and HA 1.5%-PN 1% samples had lower injection pressure than the control ( See Table 4).

In addition, in the culture medium treated with 0.025, 0.05 or 0.1% of the HA sample in human-derived fibroblasts), it was confirmed that there was no collagen synthesis ability, whereas 0.1, 0.5 or 1% of the PN sample compared to the untreated negative control group (NT) In the culture medium treated with It was confirmed that the increase was about 2.9 times and 1.8 times, respectively, compared to the control group (see FIG. 6). In addition, it was confirmed that the expression of collagen type I protein was significantly increased in the test group treated with PN 0.5%, HA 1.5%-PN 0.5% or HA 1.5%-PN 1% (see FIG. 7 ).

In addition, when the in vivo sample is administered, collagen fibers increase by sample injection in the entire tissue of the experimental group administered with HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, HA 1.5%-PN 1%, and the control group. was confirmed (see FIGS. 13 to 18). In addition, it was confirmed that the initial volume increase rate and the subsequent volume decrease rate were low compared to the control by the HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5% and HA 1.5%-PN 1% samples (Figs. 12).

In addition, if the swelling of the skin tissue is increased by the injection of the filler, pressure or pain may be caused by compressing the skin tissue. Compared to the control groups JUVEDERM VOLUMA (HA 2.0%) and JUVEDERM VOLBELLA (HA 1.5%), )-PN (1.0%) sample was injected, it was confirmed that the expression level of the protein TRPV4, the expression level of which increases when tissue swelling occurs (see FIGS. 19 to 20), is that the HA-PN complex formulation is the skin compared to the HA single formulation. It means that the skin pressure can be reduced when injected into the skin.

Therefore, the skin filler composition of the present invention has excellent physical properties compared to the control group, has excellent collagen synthesis ability in vitro or in vivo, and is expected to have less pressure and pain due to low initial swelling during injection, thus maintaining a constant shape without side effects. It can be used for medical, cosmetic, or cosmetic material purposes, and preferably can be usefully used as a filler material that can be injected into the skin soft tissue.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following examples and experimental examples are only for illustrating the present invention, and the present invention is not limited by the following examples and experimental examples.

Preparation of skin filler compositions

Samples were prepared by mixing hyaluronic acid (HA) and DNA polynucleotides (PN) having various sizes ranging from 480 to 2,000 kilodaltons (Table 1).

Specifically, after hyaluronic acid was dissolved in 0.25N sodium hydroxide at a concentration of 10% by weight, 5% of a crosslinking agent (1,4-butanediol diglycidyl ether; BDDE) was added to crosslink it in a constant temperature water bath at 50°C. The cross-linked gel is coarsely pulverized to a certain size, washed with a buffer solution, and swelled. After pulverizing the swollen gel, it is filled in a glass bottle and sterilized by heat. Salmon-derived genomic DNA was dissolved in sterile distilled water, filtered through a milliporeStericup 0.2 μm PES filter, and vacuum dried at 35° C. for 4 hours, and the pellet was dissolved in 1X PBS (Phosphate-buffered saline) to a content of 2%. Then, the crosslinked hyaluronic acid and the final concentrations of PN were mixed as shown in Table 1, respectively.

Preparation of Comparative Compositions

Same as in [Example 1], except that in [Example 1], PDRN (Polydeoxyribonucleotide) having various sizes from 1 to 195 kilodaltons (JAVENECH, France) was mixed instead of the DNA polynucleotide (PN) A mixed sample was prepared (Table 1).

Sample (wt%) Hyaluronic acid (mg/ml) DNA (mg/ml) control group HA 0.025% 0.25 - HA 0.05% 0.5 - HA 0.1% One - PN 0.1% - One PN 0.5% - 5 PN 1% - 10 Sample of Example 1 HA 1.5%-PN 0.1% 15 One HA 1.5%-PN 0.5% 15 5 HA 1.5%-PN 1% 15 10 control group HA 5%-PN 5% 50 50 HA 10%-PN 0.1% 100 One Sample of Example 2 HA 1.5%-PDRN 0.1% 15 One HA 1.5%-PDRN 0.5% 15 5 HA 1.5%-PDRN 1% 15 10 positive control JUVEDERM VOLBELLA 15 - JUVEDERM VOLUMA 20 -

<Experimental Example 1> Physical property test

<1-1> Rheological properties test

A rheometer (Kinexus, Malvern, U.K.) was used to measure the viscosity and elasticity of the samples prepared in the above Examples.

Specifically, the sample was placed between the parallel plates, and while the parallel plates were vibrating and rotating, the force to resist the force applied and the force to be lost were measured to measure the viscosity and elasticity of the sample. As a control group, JUVEDERM VOLUMA® and JUVEDERM VOLBELLA® were used and the measurement was repeated twice, and the measurement conditions were set as follows: Geometry: Pu20 S0163 SS, Oscillation mode, Shear strain: 1.5%, 0.05~25 Hz at 1 Hz Frequency, Gap: 0.5 mm, Temp. 25°C.

The shear strain in the LVER (linear viscoelastic region) was set to 1.5% by the amplitude sweep evaluation, and the viscoelastic response was measured by the frequency sweep. G'(Storage modulus; elastic modulus), G''(Loss modulus; viscous modulus), G*(Complex rigidity modulus), ŋ*(Complex viscosity), and δ(Phase angle) were measured, where G', G * is a numerical value indicating elasticity, and indicates resistance to deformation. In the case of G', it is a numerical value indicating elasticity, that is, a physical property corresponding to a solid. The larger the value, the harder the sample and the greater the ability to resist deformation. G'' is a numerical value indicating viscosity and indicates physical properties corresponding to liquids. The phase angle (δ) is a value between 0° and 90°, where a high value is G''>G', and a low value is G''<G' (Table 2). Through the above numerical values, the liquid or solid nature of the sample can be identified.

One G' (Storage modulus; elastic modulus) storage modulus,
The size of the elastic composition of the sample at the measured frequency 2 G'' (Loss modulus; viscous modulus) loss modulus,
The magnitude of the viscous composition of the sample at the measured frequency 3 Complex rigidity modulus (G*) elastic modulus(G') + viscous modules(G''): represents overall stiffness 4 ŋ*(Complex viscosity) For external forces that are both viscous and elastic
A unit for measuring the degree of deformation 5 δ (Phase angle) phase angle 6 tanδ G''/G', damping factor

As a result, as shown in Table 3 and FIGS. 1 to 3, the G' (Storage modulus) and G* (Complex modulus) values of the HA 5%-PN 5% sample among the HA and PN mixed samples were the lowest, and the G It was confirmed that the '' (Loss modulus) and δ (Phase angle) values decreased as the PN content increased. On the other hand, G' (Storage modulus), G'' (Loss modulus), G* (Complex modulus), and δ (Phase angle) values were all higher in the HA and PN mixed samples than in the control groups JUVEDERM VOLUMA® and JUVEDERM VOLBELLA®. (frequency 0.1 ~ 10Hz).

The test results show that when PN is mixed with HA, elasticity is increased, which means that resistance to external stimuli and restoration properties are increased.

In addition, in the case of the HA 10%-PN 0.1% sample with increased hyaluronic acid content, compared to the HA 1.5%-PN 0.1% sample, G' (Storage modulus), G'' (Loss modulus), G* (Complex modulus), and δ (Phase angle) were all lowered, and in the case of the HA-PN mixed formulation, it was confirmed that both the elastic properties and the viscous properties decreased as the hyaluronic acid content increased.

When PDRN of the same content as PN was mixed with hyaluronic acid, as shown in Table 3 and FIGS. 3 to 4, G' (Storage modulus), G* (Complex modulus) and G'' (Loss modulus) compared to the control group and δ (Phase angle) value increased, but was measured lower than when PN was mixed. This test result means that when PDRN is mixed with HA, elasticity is increased, but the degree is less than when PN is mixed.

Complex modulus (G*) G' (Storage modulus) G'' (Loss modulus) δ (Phase angle) ŋ*(Complex viscosity) JUVEDERM VOLUMA® 343.1238 340.9714 37.63952 6.360952 63.40595 JUVEDERM VOLBELLA® 198.9333 196.1048 32.91476 9.694286 114.9117 HA 1.5%-PN 0.1% 1197.481 1107.157 448.6429 22.84048 345.3338 HA 1.5%-PN 0.5% 1210.481 1165.652 323.5667 15.88238 360.3652 HA 1.5%-PN 1% 526.6381 509.6667 132.5667 14.59429 161.1481 HA 5%-PN 5% 500.4074 487.5238 112.819 13.83877 147.2738 HA 10%-PN 0.1% 731.0948 695.55238 225.18095 19.51294 258.6126 HA 1.5%-PDRN 0.1% 498.8666 491.54651 85.1466 10.168547 114.91611 HA 1.5%-PDRN 0.5% 516.562 506.8436 99.72883 11.12455 126.8245 HA 1.5%-PDRN 1% 520.5753 506.87712 118.63477 13.35152 131.3251

<1-2> Injection force test

In order to evaluate the pressure at the time of injection of the sample prepared in the above example, it was evaluated using a Texture Analyzer (TA.XTplus Texture Exponent Series).

Specifically, the 27G 1/2" needle was transferred to a combined syringe, mounted on a texture analysis device, and the injection speed was 15 mm/min at a constant distance. Measured under the following conditions: Temp.: 25°C, speed: 15 mm/min, distance(mm); 6.00~6.40 mm/Force(N), needle: TERUMO 27G 1/2" TW 0.4x12mm.

As a result, as shown in Table 4, it was confirmed that the HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, and HA 1.5%-PN 1% samples had lower injection pressure compared to the positive control JUVEDERM VOLUMA.

HA 1.5%-PN 0.1% HA 1.5%-PN 0.5% HA 1.5%-PN 1% JUVEDERM VOLBELLA® JUVEDERM VOLUMA® gauge 27G 15 mm/min 12.191 15.996 13.119 6.755 18.613

<Experimental Example 2> In vitro test

<2-1> Confirmation of skin wrinkle improvement effect: Measurement of collagen synthesis

The skin wrinkle improvement effect of the samples of the regular Example was investigated through Sircol Collagen assay in HDF (Human dermal fibroblast; human-derived fibroblast).

Specifically, Sircol assay kit (Biocolor Ltd., Belfast, United Kingdom) was used, and HDF was dispensed at a rate of 5 × 10 ⁴ cells/well in a 24 well-plate and cultured in DMEM with FBS for 24 hours, and FBS was After replacing it with the removed DMEM, it was cultured again for 24 hours. Thereafter, the samples prepared in Examples were treated for 72 hours at each concentration, and the supernatant of the test group and control cells was taken to measure the amount of total collagen. The amount of collagen was calculated by measuring the absorbance at 555 nm using an ELISA microplate reader (SoftMax Pro5, Molecular Devices, USA), and then creating a standard concentration curve. The treatment concentration of the sample was evaluated by selecting a concentration that does not show toxicity through a cytotoxicity test.

As a result, as shown in Figure 6, compared to the untreated negative control group (NT), it was confirmed that the collagen synthesis ability of the culture medium of the positive control group treated with vitamin C about twice. In addition, it was confirmed that there was no collagen synthesis ability in the culture medium treated with 0.025, 0.05 or 0.1% of the HA sample. On the other hand, in the culture medium treated with 0.1, 0.5 or 1% of the PN sample compared to the untreated negative control group (NT), it was confirmed that the collagen synthesis ability increased up to about 3.6 times in a concentration-dependent manner. In the culture medium treated with the HA 1.5%-PN 0.1% sample and the HA 1.5%-PN 0.5% sample, it was confirmed that the collagen synthesis capacity increased by about 2.9 times and 1.8 times, respectively, compared to the negative control group.

<2-2> Confirmation of collagen expression level at the protein level

Changes in collagen expression in the protein level of cells treated with HA and PN mixed samples were confirmed through Western blot.

HDF cells were cultured in a medium supplemented with Fetal bovine serum (FBS) for 24 hours, and samples of each concentration were treated for 72 hours in a state in which FBS was removed or FBS was included, and then cells of the test group and the control group were obtained, Western blot was performed. Specifically, the supernatant of the obtained cells was removed, washed twice with DPBS (Dulbecco's Phosphate-Buffered Saline), and then the cells were lysed using a RIPA (Radioimmunoprecipitation assay) buffer, and the cell lysate was 13,000 g at 4°C. A protein extraction supernatant from which debris was removed by centrifugation for 20 minutes was obtained. After electrophoresis of 20 μg of the quantified protein on 10% SDS-PAGE, the protein in the gel was blotted with a PVDF membrane. After blocking with 5% Skim milk, react with the primary antibody (anti-collagen Type I antibody, Cat# ab34710) at 4°C for 24 hours, and then add the secondary antibody, Goat Anti-Rabbit IgG (HRP) antibody (Cat# ab205718) to 4 The reaction was carried out at ℃ for 2 hours. After washing 3 times for 10 minutes with TBS-T, the expression level of the protein was checked using ECL detection reagents (Thermo Scientific, Pierce Biotechnology, USA).

As a result, as shown in FIG. 7 , it was confirmed that the expression of collagen type I was significantly increased in the test group treated with PN 0.5%, HA 1.5%-PN 0.5% or HA 1.5%-PN 1%.

<Experimental Example 3> In vivo test

All animal experiments used in this evaluation were performed after approval (No.201700022) by the Animal Experimental Ethics Committee operated by Chung-Ang University School of Medicine. 6-week-old SKH1-Hrhr hairless mice were purchased from Orient Bio and used. All mice were bred at a 12 hr/day light/dark cycle in a laboratory maintained in optimal environmental conditions at a temperature of 24±2° C. and a humidity of 50±10%, and food was allowed to be freely eaten. Before the experiment, a stabilization period of at least 1 week was allowed to acclimatize, and then the experiment was started.

Zoletil (30 mg/kg), an anesthetic, and Rompun (10 mg/kg), a muscle relaxant, mixed solution (3:1) were administered to the abdominal cavity of the mouse to anesthetize, and then the sample was injected into the back of the mouse. A total of 160 animals were used in 8 groups with 20 animals per group, and 5 individuals for persistence evaluation requiring evaluation of the same individual and 10 animals for tissue staining were used (Table 5). For each test group, PN 0.1%, PN 0.5%, PN 1%, HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5% or HA 1.5%-PN 1%, and a positive control group in the subcutaneous layer below the midline of the mouse HA dermal fillers, JUVEDERM VOLUMA® or JUVEDERM VOLBELLA® were injected. All samples were continuously injected by 100 μl subcutaneously, and the biocompatibility of the administered samples and Persistence was assessed ( FIG. 8 ). The experimental groups were as follows:

Group 1: PN 0.1%

Group 2: PN 0.5%

Group 3: PN 1%

Group 4: HA 1.5%-PN 0.1%

Group 5: HA 1.5%-PN 0.5%

Group 6: HA 1.5%-PN 1%

Group 7: Positive control, JUVEDERM VOLUMA (Hyaluronic acid 20 mg/ml)

Group 8: positive control, JUVEDERM VOLBELLA (Hyaluronic acid 15 mg/ml)

0'hr 1w 4w 8w 12w 18w 24w Surface topography (PrimosLITE) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) DSLR (D3200, Nikon, Japan), Folliscope (LeedM, Seoul, Korea) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) Histology (H&E, MT stain) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5) √(n=5)

<3-1> Persistence evaluation: Volumetric analysis

In order to evaluate the persistence of the sample, shape retention and mobility of the administered material using PRIMOSLITE (GFMesstechnikGmbH, Germany) and Folliscope (LeedM, Seoul, Korea) immediately after administration of the test material (0'hr) and for each evaluation period Volume changes were analyzed (n=5). Software PRIMOS 5.8 was used for the analysis program.

As shown in FIGS. 9 to 12 , when the sample was administered in vivo, as a result of magnifying it to confirm the shape and the degree of leakage, no specific details were identified. There was no significant difference in the volume immediately after administration (0'hr) for each sample, but HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, HA 1.5%-PN 1%, JUVEDERM VOLUMA® and JUVEDERM VOLBELLA All groups treated with ® showed a tendency to increase in volume up to 4 weeks and then gradually decrease thereafter. HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, and HA 1.5%-PN 1% were compared to the control JUVEDERM VOLUMA® or JUVEDERM VOLBELLA®, and it was confirmed that the initial volume increase rate and the subsequent volume decrease rate were low.

The characteristic of the positive control group HA filler is that it can cause pressure and pain during the procedure due to excessive volume increase. Therefore, HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, and HA 1.5%-PN 1% have lower initial swelling compared to JUVEDERM VOLUMA® and JUVEDERM VOLBELLA®, so pressure and pain are expected to be less. It is expected that continuous naturalness will be maintained.

<3-2> Collagen formation ability confirmation: histological analysis

The skin tissue at the sample injection site and the skin tissue not injected were extracted from the mouse and fixed in 10% neutral buffered formalin solution. Thereafter, it was embedded in paraffin and hardened, and a 5 μm section was prepared, and collagen fibers were observed with Masson's Trichrome stain. After observing the tissue slides at 100 times and 200 times using an optical microscope, the main histological characteristics of each slide were read.

As a result, as shown in FIGS. 13 to 18, in the entire tissue of the experimental group administered with HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, HA 1.5%-PN 1%, JUVEDERM VOLUMA® and JUVEDERM VOLBELLA® It was confirmed that collagen fibers increased by sample injection.

Collagen fibers are a major component of the dermal layer and are produced in fibroblasts to increase the thickness of the skin. The results show that HA 1.5%-PN 0.1%, HA 1.5%-PN 0.5%, and HA 1.5%-PN 1% induce collagen fiber production like JUVEDERM VOLUMA and JUVEDERM VOLBELLA.

<3-4> Immunofluorescence stain

It is known that the TRPV4 ion channel, which is related to temperature sensation, also responds to osmotic pressure, which is one of the mechanical stimuli. In the organum vasculosum lamina terminalis (OVLT), a site that detects the osmotic concentration of cerebrospinal fluid, the TRPV4 ion channel serves as an osmotic pressure sensing device, and other auditory hair cells, vibrissal Merkel cells, etc. cells), sensory ganglia, keratinocytes, etc. contribute to detecting changes in osmotic pressure, and are activated by detecting a decrease in osmolality of about 30 mOsm. The fact that mechanical pain was reduced in transgenic mice knocked out of the TRPV4 ion channel supports this possibility. Accordingly, in order to confirm the degree of tissue compression according to the swelling of the filler, it was attempted to confirm the change in the TRPV4 protein expressed in the tissue.

Specifically, for immunohistofluorescence staining, the skin tissue of the sample injection site and the non-injected mouse skin tissue were extracted and fixed in a 10% formalin solution. After that, it was embedded in paraffin and hardened, and a 5 μm section was prepared. Anti-TRPV4 antibody (1:200, abcam, ab39260) was used as the primary antibody, and 2.5% bovine serum albumin (BSA, Sigma, USA), After processing by mixing in a blocking buffer containing 2.5% horse serum at a ratio of 1/200, the reaction was carried out at room temperature for 4 hours. After the primary antibody reaction was completed, the tissues were washed with PBST (PBS plus 0.1% triton X-100), and FITC-goat anti-rabbit antibody (green) was added to a blocking buffer containing 2.5% BSA and 2.5% horse serum. The mixture was mixed at 1:1000 and treated in the dark for 2 hours. After washing with PBST three times, the main histological features were read using a fluorescence microscope.

As a result, as shown in FIGS. 19 and 20, only in the experimental group injected with JUVEDERM VOLUMA®, it was confirmed that TRPV4 expression of the muscle layer was stronger than that of other groups, and strong swelling in the skin tissue It was confirmed that it was affecting the surrounding tissues.

The increase in the expression of TRPV4 means that the osmotic pressure of the skin tissue is increased, and the swelling phenomenon is increased, and the swelling of the skin tissue causes compression of the tissue, thereby causing a feeling of pressure or pain. The above results show that when the formulation mixed with HA and PN is injected, the expression of TRPV4 in the muscle layer is significantly lower than that of JUVEDERM VOLUMA, which is that the mixing of PN reduces the increase in skin osmotic pressure due to filler injection, so that the skin It has been shown that pressure and pain can be reduced.

In addition, in the case of the control JUVEDERM VOLUMA having a hyaluronic acid content of 2.0%, it was confirmed that the expression of TRPV4 was stronger than that of the control JUVEDERM VOLBELLA having a hyaluronic acid content of 1.5% and the test sample HA 1.5%-PN 1%. This means that JUVEDERM VOLUMA presses the tissue more strongly, and the higher the hyaluronic acid content, the stronger the tissue compression caused by the swelling of the skin tissue.

Claims (6)

A dermal filler composition for tissue repair comprising hyaluronic acid or a salt thereof and a polynucleotide as active ingredients,
The molecular weight of the polynucleotide is 480 to 2,000 kilodaltons (kDa),
It is included in 0.1 to 0.5% by weight of the total weight of the composition,
The hyaluronic acid is a cross-linked hyaluronic acid having a molecular weight of 500 to 6000 kilodaltons,
The cross-linked hyaluronic acid is included in 1.5% by weight of the total weight of the composition,
The dermal filler composition for tissue repair has a storage modulus of 1100 Pa or more and a complex viscosity of 300 Pa·s or more.
delete delete The dermal filler composition for tissue repair according to claim 1, wherein the composition increases the synthesis of collagen.
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