KR20200023613A - Nano hydrogel for filler procedure with 3 dimension network structure using exosome surface protein and use thereof - Google Patents

Abstract

Provided is a nano hydrogel for filler procedure with improved biocompatibility, comprising exosomes derived from stem cell culture. The nano hydrogel is a hydrogel formed of a polymer main chain, a crosslinking agent, and an exosome, wherein the crosslinking agent is at least partially in a combined state with the exosome.

Description

NANO HYDROGEL FOR FILLER PROCEDURE WITH 3 DIMENSION NETWORK STRUCTURE USING EXOSOME SURFACE PROTEIN AND USE THEREOF}

The present invention relates to a nano hydrogel for filler treatment in which a three-dimensional network structure is formed using an exosome surface protein, and a use thereof.

In another aspect, the present invention relates to a nano hydrogel for filler procedures including improved exosomes derived from stem cell culture. In detail, the present invention relates to a nano hydrogel for filler treatment in which exosomes derived from stem cell culture and a polymer backbone are combined to improve physical properties. More specifically, the present invention relates to a nano hydrogel for filler treatment that can be controlled to nano size by improving physical properties including exosomes, thereby improving water content and biocompatibility.

Filler treatment is a procedure used for cosmetic purposes such as correcting the function of the human body by injecting a dermal filler under the skin, mainly into the dermal layer, or removing wrinkles on the face. It is possible to obtain a surgical effect by a simple procedure without plastic surgery, and the treatment time is short and everyday life is immediately possible, which is attracting attention from busy modern people.

Skin fillers can be roughly classified into first-generation fillers using animal collagen or human collagen and second-generation fillers using hyaluronic acid.

First-generation fillers are rarely used in recent years because of their short duration and the hassle of performing skin hypersensitivity tests for the procedure.

Hyaluronic acid filler, the second-generation filler, is composed of polysaccharides, N-acetyl-D-glucosamine and D-glucuronic acid, which have a longer duration than collagen fillers and are similar to human components, and have fewer side effects such as skin hypersensitivity reactions. It is easy to remove and can attract water to maintain skin moisture, maintain volume and elasticity, and thus have suitable advantages as a filler for skin.

Since hyaluronic acid fillers generally have an average size of about 30 μm to 700 μm, it is common to inject into the skin or subcutaneous fat using a syringe needle. However, the injection method is difficult to control the insertion position, insertion depth, etc. of the needle has a side effect such as damaging important blood vessels, nerves, etc. or filler is injected into the blood vessel during the procedure. In particular, since many blood vessels are distributed around the eye, filler material flows into the arteries during the procedure of orbit around the eye, blocking eye arteries or cerebrovascular vessels, which may cause serious side effects such as skin necrosis, cerebral infarction, and blindness.

On the other hand, since hyaluronic acid is easily decomposed, hyaluronic acid fillers that have been cross-linked or cross-linked by adding a crosslinking agent to hyaluronic acid have been developed in order to increase the retention period. Such hyaluronic acid fillers vary in physical properties such as viscosity, elasticity, and stability of the filler depending on the crosslinking rate (or crosslinking rate). When the crosslinking rate is low, the elasticity is low, and in this case, the injection form may not be maintained for a long time and the holding period may be short. On the other hand, when the crosslinking rate is high, the elasticity is high and the filler form during injection is maintained for a long time, but the possibility of leaving the injection site increases. In addition, if the crosslinking rate is extremely high, it may not be decomposed in the human body, and thus rejection, film formation, and granulomatous formation may occur.

Thus, there is a need for a hyaluronic acid-based filler having a high level of stability and having an appropriate level of viscosity and elasticity.

 A Review of the Metabolism of 1,4-Butanediol Diglycidyl Ether-Crosslinked Hyaluronic Acid Dermal Fillers, Koenraad de Boulle et al., Dermatologic surgery 2013, 1758-1766 (Non-Patent Document 2) Study of the effect of mixing approach on cross- linking efficiency of hyaluronic acid-based hydrogel cross-linked with 1,4-butandiol diglycidyl ether, Mohammed Al-Sibani et al. (2016) (Non-Patent Document 3) Comparative Physical Properties of Hyaluronic Acid Dermal Fillers, Jeffrey Kablik et al., Dermatologic surgery 2009, 302-312

Non-Patent Document 1 reports on a method of increasing physical properties of hyaluronic acid hydrogel, which is most commonly used in the past. Non-Patent Document 1 discloses a hydrogel using a hyaluronic acid polymer and 1,4-butanediol diglycidyl ether (1,4-Butanediol diglycidyl ether, BDDE). In addition, Non Patent Literature 2 and Non Patent Literature 3 relate to physical properties of hyaluronic acid hydrogel, and Non Patent Literature 2 and Non Patent Literature 3 report that the hydrogel polymer physical properties decrease depending on the crosslinking conditions.

As such, the physical properties such as viscosity, elasticity and biocompatibility of the hydrogel are very important factors in the hydrogel for filler treatment. However, it is difficult to control the size of the hydrogel in order to maintain suitable physical properties of the hydrogel. That is, when reducing the size of the hydrogel for filler treatment does not exhibit a level of physical properties suitable for the filler procedure, on the other hand, there is a limit that the hydrogel particles have a micro level size when maintaining the physical properties of the hydrogel to be suitable for the filler procedure. Accordingly, the procedure can only be performed using a syringe needle as in the prior art.

Therefore, the problem to be solved by the present invention is to provide a nano-hydraulic gel for filler treatment that can exhibit a level of physical properties suitable for filler procedures for humans despite the size control to have a nano-size. In addition, to provide a nano hydrogel that can have an excellent biocompatibility can reduce side effects.

At the same time, it is to provide a nano hydrogel for filler treatment that can be applied to the surface of the skin and not induced by subcutaneous injection with nano-sized, to induce natural absorption.

Another object of the present invention is to provide a method for preparing a nano hydrogel for filler treatment having excellent physical properties and biocompatibility.

Another problem to be solved by the present invention is to provide a filler composition suitable for application to the skin.

The objects of the present invention are not limited to the above-mentioned technical problems, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

Nano hydrogel for filler treatment according to an embodiment of the present invention for solving the above problems is a hydrogel formed of a polymer backbone, a crosslinking agent and an exosome, the crosslinking agent is at least partly bonded to the exosomes.

The polymer backbone may include a hyaluronic acid backbone.

Here, the crosslinking agent may include polyethylene glycol dimethacrylate. In some embodiments, the crosslinking agent may consist solely of polyethylene glycol dimethacrylate.

In addition, the exosomes may comprise HEK293T.

At least a part of the crosslinking agent may crosslink the hyaluronic acid backbone and the surface protein of the exosome to form a network structure in which the exosome is homogeneously distributed.

In addition, the molecular weight of the polyethylene glycol dimethacrylate single molecule may be 500 g / mol or less. When the molecular weight of the crosslinking agent is too large as in Experimental Example 1 to be described later, the physical properties of the hydrogel may decrease.

The average particle size of the nano hydrogel is in the range of 10nm to 600nm, the viscosity of the nano hydrogel (@ 27 ℃, 1Hz rheometer) may be in the range of 30Pa · s to 100Pa · s.

At the same time, the filler hydro nanogel is applied to the skin surface without being injected through the subcutaneous skin, the skin penetration rate of the nano hydrogel may be 70 μg / cm ² · 24h or more.

In some embodiments, the nano hydrogel for filler treatment, containing an aqueous solution of hyaluronic acid having a concentration of 30mg / ml to 40mg / ml, 0.1wt% to 8.0wt% bifunctional crosslinking agent and HEK293T compared to the aqueous hyaluronic acid solution As a stem cell culture medium, 0.01 wt% to 1.0 wt% of the stem cell culture solution is mixed with respect to the aqueous hyaluronic acid solution, the mixture is aged at 45 ° C. to 50 ° C. for at least 24 hours, and the aged mixture at 30 ° C. to 35 ° C. It is prepared by reacting with stirring.

Method for producing a nano hydrogel according to an embodiment of the present invention for solving the other problems, the aqueous solution of hyaluronic acid having a concentration of 30mg / ml to 40mg / ml, 0.1wt% to 8.0wt% of the hyaluronic acid aqueous solution As a stem cell culture medium containing a bifunctional crosslinking agent and HEK293T, 0.01% to 1.0% by weight of the stem cell culture solution is mixed with respect to the aqueous hyaluronic acid solution, and the mixture is aged at 45 ° C to 50 ° C for at least 24 hours, and the aging The prepared mixture is prepared by reacting with stirring at 30 ° C to 35 ° C.

Filler composition according to an embodiment of the present invention for solving the above another problem includes a nano hydrogel according to the present invention described above.

Details of other embodiments are included in the detailed description.

According to embodiments of the present invention, a nano hydrogel comprising a exosome derived from stem cells and having a stable three-dimensional network structure may be provided by combining a polymer backbone such as a hyaluronic acid backbone with the exosome.

Therefore, despite having a nano-size, it is possible to maintain excellent viscosity and elasticity and to improve biocompatibility derived from components such as phospholipids contained in exosomes. In addition, since the hydrogel has a nano size, it can be induced in the body by applying it to the skin surface instead of being treated through subcutaneous injection, and can minimize side effects due to filler treatment.

Effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

1 is a schematic diagram showing a unit of a nano hydrogel according to an embodiment of the present invention.
Figure 2 is a schematic diagram showing the exosomes of Figure 1.
Figure 3 is a schematic diagram showing the binding between the hyaluronic acid backbone and exosomes of FIG.
Figure 4 is a schematic diagram showing a state in which the crosslinking agent of Figure 1 is crosslinked with the hyaluronic acid backbone and exosomes.
5 is a flowchart illustrating a method of manufacturing a nano hydrogel according to an embodiment of the present invention.

Advantages and features of the present invention, and methods for achieving them will be apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, only the embodiments are to make the disclosure of the present invention complete, and those of ordinary skill in the art to which the present invention belongs. It is provided to fully inform the scope of the invention, and the invention is defined only by the scope of the claims.

That is, various changes may be made to the embodiments proposed by the present invention. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, 'and / or' includes each and all combinations of one or more of the items mentioned. In addition, the singular also includes the plural unless specifically stated otherwise in the text.

As used herein, 'comprises' and / or 'comprising' does not exclude the presence or addition of one or more other components in addition to the components mentioned. Numerical ranges shown using 'to' indicate numerical ranges including the values described before and after the lower limit and the upper limit, respectively. "About" or "approximately" means a value or numerical range within 20% of the value or numerical range described thereafter.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification are used in a sense that can be commonly understood by those skilled in the art. In addition, terms that are defined in a commonly used dictionary are not ideally or excessively interpreted unless they are clearly defined in particular.

In the present specification, unless otherwise stated, 'molecular weight' means 'weight-average molecular weight' and the unit uses g / mol. In addition, unless otherwise indicated in this specification, the unit regarding temperature is represented by "degreeC."

Hereinafter, the present invention will be described in detail.

1 is a schematic diagram showing a unit of a nano hydrogel according to an embodiment of the present invention. Figure 2 is a schematic diagram showing the exosomes of Figure 1.

Referring to FIGS. 1 and 2, the nano hydrogel 10 for the filler procedure according to the present embodiment may include a polymer backbone 100, a crosslinking agent or a crosslinking chain 200, and an exosome 300.

The polymer backbone 100 is a stem molecular chain constituting the skeleton of the polymer, and in order to be used as a filler composition, it should be physiologically harmless to the human body and stable. Polymer main chain 100 according to an embodiment of the present invention may be a hyaluronic acid backbone. Hereinafter, the hyaluronic acid backbone is described as an example of the polymer backbone 100, but the present invention is not limited thereto. Other polymer backbones known as hydrogels for filler treatment may also be used.

The crosslinking agent 200 is capable of crosslinking the hyaluronic acid backbone 100 and the exosome 300 through a chemical bond, and includes dimethacrylates, alkyldiepoxys, diglycidyl ethers, and divinyl sulfones. And epichlorohydrin. The crosslinking agent may be a bifunctional compound having a pair of functional groups capable of reacting with the hyaluronic acid backbone 100 and the exosome 300.

In an exemplary embodiment, the crosslinking agent 200 is polyethylene glycol dimethacrylate (PEGDMA), 1,4-butanediol diglycidyl ether (1,4-Butanediol diglycidyl ether, BDDE) 1 1,2,7,8-Diepoxyoctane, 1,5-hexadiene diepoxide and bisphenol A diglycidylether It may comprise one or more selected from the group consisting of. Preferably, the crosslinking agent 200 may include polyethylene glycol dimethacrylate. More preferably, the crosslinking agent 200 may be composed of only polyethylene glycol dimethacrylate. The inventors of the present invention have confirmed that no binding occurs with the exosome 300, which will be described below, for some of the known crosslinking agents, and completed the present invention. This will be described later together with an experimental example.

Exosome 300 is a micro-vesicle (endoves) originated from the endosomes (endosome) (micro-vesicle) (vesicles of tens to hundreds of nanometers in size consisting of the same phospholipid membrane of the cell membrane structure. Exosomes 300 are membrane endoplasmic reticulum secreted from various types of cells. For example, exosomes 300 are released or secreted from cells of various animals, plants, bacteria, fungi, algae, etc., cell cultures, cell culture supernatants, stem cell cultures, stems Dispersed, suspended, precipitated, suspended or mixed in biological solutions such as cell culture supernatants, whole blood, serum, umbilical cord blood, plasma, ascites fluid, brain and cerebrospinal fluid, placental extract and bone marrow aspirate.

In an exemplary embodiment, the exosomes 300 according to the present invention may be vesicles secreted and released from stem cells. More preferably, the exosomes 300 may be HEK293T cells, which are human embryonic kidney (Human Embryonic Kidney) cell lines, but the present invention is not limited thereto.

HEK293T cells are composed of a double phospholipid membrane consisting of a hydrophilic head 310 made of phosphate groups and a hydrophobic tail 320 made of fatty acids, and relatively less genetic material such as DNA and RNA than other exosomes. Almost empty

Hereinafter, the structure of the nano hydrogel for filler treatment according to the present embodiment will be described in more detail with reference to FIGS. 3 and 4.

Figure 3 is a schematic diagram showing the binding between the hyaluronic acid backbone of Figure 1 and the exosomes. Figure 4 is a schematic diagram showing a state in which the crosslinking agent of Figure 1 is crosslinked with the hyaluronic acid backbone and exosomes.

3 and 4, the exosomes and hyaluronic acid may be connected by PEGDMA crosslinking agent to form a nano hydrogel having a three-dimensional network structure. Specifically, methacrylate groups located at both ends of PEGDMA may bind to the hydroxy group of the protein and hyaluronic acid backbone of the surface of the exosome, respectively, or both of the methacrylate groups may bind to the hyaluronic acid backbone. That is, at least some of the crosslinking agent crosslinks the hyaluronic acid backbone and the protein on the surface of the exosome to form an exosome-PEGDMA-hyaluronic acid backbone crosslinker or a hyaluronic acid-PEGDMA-hyaluronic acid backbone crosslinker, whereby the exosomes are homogeneous. The nano hydrogel of the three-dimensional network structure can be formed can be formed.

3 and 4 illustrate the present invention by illustrating a specific chemical structure of a hyaluronic acid polymer chain and a PEGDMA crosslinking agent, but the present invention is not limited thereto.

Most hyaluronic acid hydrates prepared through chemical crosslinking between the hyaluronic acid backbone and the crosslinking agent have particle sizes ranging from several microns to several tens of microns, making it difficult to form hyaluronic acid hydrate nanoparticles. In addition, conventionally, even when nanoparticles are formed, it is difficult to maintain a three-dimensional network structure, and properties such as viscosity and elasticity are not good.

On the other hand, the nano hydrogel for filler treatment according to the present invention increases the binding strength between the hyaluronic acid backbone by adding an exosome between the hyaluronic acid backbone, thereby maintaining a three-dimensional network structure, thereby ensuring excellent properties such as viscosity and elasticity Can be.

For example, the viscosity of the nano hydrogel according to this embodiment is about 30 Pa.s or more, or about 40 Pa.s or more, or about 50 Pa.s or more, or about 60 Pa.s or more, or about 70 Pa.s or more, or about It may have high viscosity properties of 80 Pa · s or more. The upper limit of the viscosity is not particularly limited, but may be preferably about 150 Pa · s or less in consideration of the bio-injection characteristics and the use as a filler.

In addition, the elasticity of the nano hydrogel according to this embodiment is about 400Pa or more, or about 450Pa or more, or about 500Pa or more, or about 550Pa or more, or about 600Pa or more, or about 650Pa or more, or about 700Pa or more, or about 750Pa or more A high elasticity of about 800 Pa or more, or about 850 Pa or more may also have physical properties. The upper limit of the elasticity is not particularly limited, but may be preferably about 1,000 Pa or less in consideration of the bio-injection characteristics and the use as a filler.

The nano hydrogel for filler treatment of the present invention may exhibit improved skin absorption ability compared to microparticles. In other words, as the particle size becomes smaller, the cosmetic disease can be treated in a non-invasive manner. For example, it may be applied to the skin to alleviate wrinkles or wrinkles of the skin (eg, facial wrinkles and facial wrinkles), glabellar wrinkles, nasolabial folds, chin wrinkles, marionette wrinkles, perioral wrinkles, and wrinkles around the eyes.

For example, the above skin penetration rate of the hydrogel is from about nano 50㎍ / cm ² · 24h according to the present embodiment, or about 60㎍ / cm ² · 24h or more, or about 70㎍ / cm ² · 24h or more, or about 80 ㎍ / cm ² · 24h may have, or at least about 90㎍ / cm ² · 24h or more, or about 100㎍ / cm ² · 24h or more high skin permeability. In the present specification, the unit 'μg / cm ² · 24h' means the weight (μg) penetrating about 1 cm ² for 24 hours. The upper limit of the skin penetration rate is not particularly limited, but may be preferably about 150 µg / cm ² · 24h or less considering the use as a filler and the effect of improving wrinkles.

Hereinafter, a method for preparing a nano hydrogel for filler treatment according to the present invention will be described. However, redundant descriptions of components substantially the same as or similar to those described above will be omitted, which will be clearly understood by those skilled in the art.

5 is a flowchart illustrating a method of manufacturing a nano hydrogel according to an embodiment of the present invention.

Referring to FIG. 5, in the method of preparing a nano hydrogel for filler treatment according to an embodiment of the present invention, preparing a mixture of a hyaluronic acid aqueous solution, a bifunctional crosslinking agent, and a stem cell culture solution (S100), and the mixture It may include the step of aging (S200) and the step of reacting while stirring the aged mixture (S300).

Specifically, the step of preparing a mixture (S100) is preparing a hyaluronic acid aqueous solution (S110), preparing a bifunctional crosslinking agent (S120), preparing a stem cell culture medium containing an exosome (S130) and mixing them It includes.

In an exemplary embodiment, the concentration of the aqueous hyaluronic acid solution may be used 30mg / mL to 40mg / mL, the amount of the bifunctional crosslinking agent may be 0.1wt% to 8.0wt% compared to the aqueous hyaluronic acid solution, polyethylene glycol dimethacryl Although rates can be used, it is a matter of course that the present invention is not so limited.

In addition, the added amount of the stem cell culture solution may be 0.01wt% to 1.0wt% compared to the aqueous hyaluronic acid solution, the exosome may use HEK293T, but the present invention is not limited thereto, and the type of crosslinking agent, exosomes, etc. Those skilled in the art will clearly understand that known ones may be used.

However, the inventors of the present invention have confirmed that the nano hydrogel according to the present embodiment has excellent efficacy under certain combinations, and thus came to complete the present invention. This will be described later in detail together with an experimental example.

Next, the method may further include a step (S200) of aging the mixture obtained in the step (S100). Exemplary embodiments of the present invention can increase the physical properties such as viscosity, elasticity of the nano hydrogel by adding the aging step (S200). This will be described later in detail together with an experimental example.

Aging may be performed at about 45 ° C. to about 50 ° C. for at least 24 hours. If the aging time is less than 24 hours, the effect of aging is insignificant and it may not be able to achieve the increase of physical properties to achieve through aging. In addition, if the aging temperature is less than 45 ℃ or exceeds 50 ℃ it may not be able to achieve the purpose of aging rather as will be described later.

Next, a step of reacting the aged mixture obtained in the step (S200) at about 30 ℃ to about 35 ℃. The stirring reaction may use a homogenizer or the like, but the present invention is not limited thereto.

The lower limit of the stirring speed is not particularly limited but may be about 500 rmm or more in terms of efficient stirring. On the other hand, if the stirring speed is too large, the formed nano hydrogel may be pulverized or efficient crosslinking may not be achieved. The upper limit of the stirring speed may be about 1,000 rpm. In addition, the preparation of the hydrogel may use a known technique, so a detailed description thereof will be omitted.

Hereinafter, the present invention will be described in more detail with reference to specific production examples, comparative examples, and experimental examples. However, the experimental examples described below are only for emphasizing the specific effects of the present invention and for illustrating the configuration of the present invention, and the scope of the present invention should not be understood as being limited thereto.

Comparative Example 1 Preparation of Hyaluronic Acid Hydrogel Without Mixing Exosomes

About 400 kDa of hyaluronic acid 35 mg / mL was added to 1 L of distilled water and stirred at 500 rpm in a vacuum to form an aqueous hyaluronic acid solution. And 0.3 wt% of polyethylene glycol dimethacrylate (PEGDMA) was added as a crosslinking agent, and it mixed. The content of the crosslinker was calculated as a percentage of the total weight of the mixture.

And while maintaining the prepared mixture at 35 ℃ stirred and reaction at 500rpm to prepare a cross-linked hyaluronic acid hydrogel. The crosslinking time of the hydrogel was properly adjusted by examining the progress of crosslinking. Other crosslinking conditions and agitation times were appropriately adjusted so that the final hydrogel had a nano size particle size.

Comparative Example 2 to Comparative Example 5 Preparation of Hyaluronic Acid Hydrogel Using HEK293T

Stem cell cultures containing HEK293T were prepared. The culture medium is that the concentration of the human embryonic kidney cell line HEK293T cells DMEM (Dulbecco's Modified Eagle Medium) with the culture medium 5% CO _2/24 hours at conditions of 37 ℃ and incubated for 10 at a low temperature filter as a filter by collecting them fold Used.

Further, about 500 kDa of hyaluronic acid 35 mg / mL was added to 1 L of distilled water and stirred at 500 rpm in a vacuum state to form an aqueous hyaluronic acid solution. And 0.3 wt% cross-linking agent described in Table 1 and 0.5wt% of the prepared culture solution was added. The content of the crosslinking agent and the culture solution was calculated as a ratio with respect to the total weight of the mixture.

Then, the prepared mixture was stirred while maintaining at 35 ° C. for a predetermined time to prepare a crosslinked hyaluronic acid hydrogel. Other crosslinking conditions and time were appropriately adjusted to observe the progress of crosslinking and the final hydrogel reacted well (Comparative Example 2 to Comparative Example 5).

Polymer Exosomes Crosslinking agent Comparative Example 2 HA (hyaluronic acid) HEK293T 1,4-butanediol diglycidyl ether Comparative Example 3 HA (hyaluronic acid) HEK293T 1,4-bisglycidyloxybutane Comparative Example 4 HA (hyaluronic acid) HEK293T 1,2,7,8-diepoxyoctane Comparative Example 5 HA (hyaluronic acid) HEK293T 1,5-hexadiene diepoxide

Preparation Examples 1 to 3 Preparation of Hyaluronic Acid Hydrogel Using HEK293T

A hydrogel was prepared in the same manner as in Comparative Example 2 except that polyethylene glycol dimethacrylate (PEGDMA) was used as a crosslinking agent. At this time, the monomolecular weight of polyethylene glycol dimethacrylate was used as shown in Table 2 below. In addition, the crosslinking conditions and time were appropriately adjusted to observe the progress of crosslinking and the final hydrogel reacts well.

PEGDMA molecular weight Preparation Example 1 300 Preparation Example 2 500 Preparation Example 3 700

<Experimental Example 1 Viscosity, Elasticity, and Strain Measurement of Comparative Examples 1 to 5 and Preparation Examples 1 to 3>

The physical properties of the hydrogels according to Comparative Examples 1 to 5, and Preparation Examples 1 to 3 prepared above were measured. Specifically, viscosity, elasticity, and strain were measured.

Viscosity was measured under Shear rate 1s ^-1 , 27 ° C, and elasticity was measured under Frequency 1 Hz, 27 ° C.

The degree of modification (MOD) is compared to the number-of-moles of N-acetyl-D-glucosamine in the unit unit of hyaluronic acid contained in the polymer hyaluronic acid (N-acetyl-D-glucosamine (GlcNAc) + D-glucuronic acid). It is the numerical value which shows the number-of-moles of the crosslinking agent couple | bonded with the whole hyaluronic acid molecule. Strain was measured by nuclear magnetic resonance spectroscopy (NMR) and performed under 30 ° pulse, 512 scans (5s relaxation delay) conditions using FT-NMR system.

The measured viscosity, elasticity and strain results are shown in Table 3 below.

Viscosity (Pas) Elasticity (Pa) % Strain Comparative Example 1 5.5 157.4 6.4 Comparative Example 2 10.6 358.5 7.5 Comparative Example 3 12.2 247.2 8.0 Comparative Example 4 8.4 346.4 6.4 Comparative Example 5 21.3 378.2 9.8 Preparation Example 1 35.2 497.4 3.8 Preparation Example 2 42.1 587.5 3.4 Preparation Example 3 30.5 488.6 5.1

Referring to Table 3, it can be seen that in the case of Comparative Example 1 without crosslinking the exosomes, the viscosity and elasticity are very low. In particular, it was confirmed that the viscosity was too low to maintain the shape of the gel is not suitable for the filler procedure.

Comparing Comparative Example 2 to Comparative Example 5 and Preparation Examples 1 to 3, Comparative Examples were confirmed that the viscosity and elasticity is generally poor. On the other hand, in the case of Preparation Examples 1 to 3 it was confirmed that the relatively excellent physical properties. In addition, compared to the comparative examples, it can be seen that the preparations show a low strain rate despite the excellent physical properties, which can provide a filler exhibiting excellent physical properties even when a small amount of crosslinking agent is used in preparing the filler. This means that it has been improved.

Although the present invention is not limited to any theory, the crosslinking agents used in Comparative Examples 2 to 5 did not form crosslinks with HEK293T, whereas the polyethyleneglycol dimethacrylate used in Preparation Examples 1 to 3 was compared with HEK293T. It is assumed that some crosslinking was formed.

In addition, in Preparation Examples 1 to 3, it can be confirmed that in the case of Preparation Example 1 and Preparation Example 2 using a crosslinking agent having a relatively small molecular weight, it has relatively excellent physical properties compared to Preparation Example 3. Although the present invention is not limited to any theory, it is presumed that exosomes do not contribute to the improvement of physical properties of the hydrogel when the length of the chain of polyethylene glycol dimethacrylate is too long.

Experimental Example 2: Measurement of Particle Size of Hydrogel

The particle size of the hydrogel according to Comparative Examples 1 to 5, and Preparation Examples 1 to 3 prepared above was measured, and the particle size was measured by a particle size analyzer (Particle Size Analyzer, PSA) (Mastersizer 2000, Malvern Instrument, UK). Confirmed. And the results are shown in Table 4 below.

Average particle size (nm) peak Comparative Example 1 612 1 place Comparative Example 2 1,243 1 place Comparative Example 3 915 2 places Comparative Example 4 854 2 places Comparative Example 5 1065 1 place Preparation Example 1 697 1 place Preparation Example 2 734 1 place Preparation Example 3 641 2 places

Referring to Table 4, in the case of Comparative Example 3 and Comparative Example 4 it can be confirmed that the particle size control is very difficult. Moreover, although the manufacture example 3 was also weak, it was confirmed that two peaks appeared.

In Comparative Examples 2 and 5, it was confirmed that the average particle size was about 1,000 nm or more. On the other hand, in the case of Comparative Example 1, Preparation Example 1 and Preparation Example 2 was confirmed that the particle size control is well because there is one peak. In addition, the average particle size was confirmed that the level of about 400nm to 600nm.

<Comparative Example 6 to Comparative Example 8: Exosome content change>

A hydrogel was prepared in the same manner as in Preparation Example 2, except that the content of the stem cell culture solution was changed (increased) as in Table 5 below. The content of the stem cell culture was calculated as a ratio to the total weight of the mixture.

Stem cell culture content (wt%) Comparative Example 6 1.5 Comparative Example 7 2.0 Comparative Example 8 3.0

Preparation Example 4 and Preparation Example 5 Exosome Content Change>

A hydrogel was prepared in the same manner as in Preparation Example 2, except that the content of the stem cell culture was changed as shown in Table 6 below. The content of the stem cell culture was calculated as a ratio to the total weight of the mixture.

Stem cell culture content (wt%) Preparation Example 4 0.1 Preparation Example 5 1.0

Experimental Example 3 Measurement of Viscosity, Elasticity, and Strain of Hydrogel

The physical properties of the hydrogels according to Preparation Example 2, Preparation Example 4, Preparation Example 5 and Comparative Examples 6 to 8 prepared above were measured. Viscosity, elasticity, and strain were measured in the same manner as in Experimental Example 1. And the results are shown in Table 7 below.

Viscosity (Pas) Elasticity (Pa) % Strain Preparation Example 2 42.1 587.5 3.4 Preparation Example 4 43.4 605.4 3.5 Preparation Example 5 45.5 565.1 3.4 Comparative Example 6 35.7 554.7 5.1 Comparative Example 7 30.8 478.4 6.4 Comparative Example 8 30.2 453.1 8.6

Referring to Table 7, it can be seen that in the case of Preparation Example 4 and Preparation Example 5 exhibits the same or more physical properties as Preparation Example 2. On the other hand, in the case of Comparative Examples 6 to 8 it can be seen that the physical properties are inferior as in Preparation Example 2.

In addition, while Preparation Example 4 and Preparation Example 5 had the same level of strain as Preparation Example 2, it can be seen that the Comparative Example 6 to Comparative Example 8 has a higher strain than the Preparation Example 2.

Preparation Example 6 Preparation of Hydrogel by Adding Aging Step

Hydration gel was prepared in the same manner as in Preparation Example 2, except that the aqueous hyaluronic acid solution, the crosslinking agent, and the stem cell culture were mixed and then aged for 30 hours in a dark room at 45 ° C.

Preparation Example 7 Preparation of Hydrogel by Adding Aging Step

A hydrogel was prepared in the same manner as in Preparation Example 2, except that an aqueous hyaluronic acid solution, a crosslinking agent, and a stem cell culture were mixed and then aged for 24 hours in a dark room at 50 ° C.

Comparative Example 9: Preparation of Hydrogel by Adding Aging Step

A hydrogel was prepared in the same manner as in Preparation Example 2, except that an aqueous hyaluronic acid solution, a crosslinking agent, and a stem cell culture were mixed and then aged for 48 hours in a dark room at 30 ° C.

Aging conditions of Preparation Example 6, Preparation Example 7 and Comparative Example 9 are summarized in Table 8 below.

Temperature (℃) Hours (h) Preparation Example 6 45 30 Preparation Example 7 50 24 Comparative Example 9 30 48

Experimental Example 4 Measurement of Viscosity, Elasticity, and Strain of Hydrogel

The physical properties of the hydrogels according to Preparation Example 2, Preparation Example 6, Preparation Example 7 and Comparative Example 9 were prepared. The viscosity, elasticity, and strain were measured in the same manner as in Experimental Example 1. And the results are shown in Table 9 below.

Viscosity (Pas) Elasticity (Pa) % Strain Preparation Example 2 42.1 587.5 3.4 Preparation Example 6 84.4 815.3 3.0 Preparation Example 7 79.4 794.0 2.9 Comparative Example 9 44.9 580.1 3.6

Experimental Example 5: Measurement of Particle Size of Hydrogel

The particle size of the hydrogels according to Preparation Example 2, Preparation Example 6, Preparation Example 7, and Comparative Example 9 were measured. The particle size was measured in the same manner as in Experimental Example 2. And the results are shown in Table 10 below.

Average particle size (nm) peak Preparation Example 2 734 1 place Preparation Example 6 610 1 place Preparation Example 7 578 1 place Comparative Example 9 756 2 places

Referring to Tables 9 and 10, it can be seen that in the case of Preparation Example 6 and Preparation Example 7, the viscosity and elasticity are significantly increased. In addition, in the case of Preparation Example 6 and Preparation Example 7 it was confirmed that the mean particle size is smaller than the preparation Example 2 to a significant degree. That is, before performing the main reaction between the HEK293T and the crosslinking agent in the stem cell culture, it can be seen that the preliminary reaction for a sufficient time to achieve the effect of increasing the physical properties such as viscosity, elasticity and decrease the average particle size.

On the other hand, in the case of Comparative Example 9 was confirmed to exhibit substantially the same physical properties and particle size as Preparation Example 2. That is, when the temperature of the aging is too low or the time of aging is too long, it can be seen that there is substantially no effect on the increase in physical properties and the particle size control.

The present invention is not limited to any theory, but the aging step increases the entanglement between hyaluronic acid main chains, thereby increasing the crosslinking between different chains rather than crosslinking in the same chain, and furthermore, the binding force between the crosslinking agent and the exosome. It is presumed that the physical properties have been improved.

Experimental Example 6: Measurement of Skin Absorption Rate

The skin absorption test of the hydrogels according to Preparation Example 2, Preparation Example 6 and Preparation Example 7 prepared above was carried out.

Eight weeks old male hairless guinea pigs (strain IAF / HA-hrBP) were used for the skin absorption experiment. The skin of the guinea abdomen was cut out and mounted in Frans-type diffusion cells (lab fine instruments). Distilled water was put into a receptor vessel (5 ml) of a Franz-type permeate cell vessel, and the permeate cell was mixed and dispersed at 6,000 rpm while maintaining 32 ° C., and the hydration gel according to Preparation Examples 2, 6 and 7 Each 200 μl of the solution was put in a donor container.

Absorption and diffusion were carried out according to a predetermined time, and the area of skin where absorption and diffusion occurred was 0.64 cm ² . After the absorption and diffusion of the hydrogel is finished, remove residues from the surface of the skin with 10 µl of distilled water, and use a tip type equalizer (Polytron PT2100, Switzerland). Filtration was carried out using a filtration membrane and the content was measured by high pressure liquid chromatography. And the results are shown in Table 11 below.

Absorption amount (㎍ / ㎠) Preparation Example 2 73 Preparation Example 6 121 Preparation Example 7 135

Referring to Table 11, it can be seen that the hydrogels according to Preparation Example 6 and Preparation Example 7 exhibit a much higher level of skin absorption or penetration than the hydrogel according to Preparation Example 2. This may be because the hydrogels according to Preparation Examples 6 and 7 have a very small size while maintaining excellent physical properties, but the present invention is not limited thereto.

Although described above with reference to the embodiments of the present invention, which is merely an example and not limiting the present invention, those skilled in the art to which the present invention pertains without departing from the essential characteristics of the embodiments of the present invention. It will be appreciated that various modifications and applications not illustrated above are possible.

Therefore, the scope of the present invention should be understood to include modifications, equivalents, or substitutes of the technical spirit exemplified above. For example, each component specifically shown in the embodiment of the present invention may be modified. And differences relating to these modifications and applications will have to be construed as being included in the scope of the invention defined in the appended claims.

10: hydrogel
100: hyaluronic acid backbone
200: crosslinking agent
300: exo

Claims (7)

A hydrogel formed of a polymer backbone, a crosslinking agent and an exosome, wherein the crosslinking agent is at least partly bonded with the exosome. The method of claim 1,
The polymer backbone includes a hyaluronic acid backbone, the crosslinking agent includes polyethylene glycol dimethacrylate, and the exosomes include HEK293T. The method of claim 2,
At least some of the crosslinking agent crosslinks the hyaluronic acid backbone and the surface protein of the exosome to form a network structure in which the exosome is homogeneously distributed, the nano hydrogel for filler treatment. The method of claim 2,
The molecular weight of the polyethylene glycol dimethacrylate single molecule is 500g / mol or less nano hydrogel for filler treatment. The method of claim 1,
The average particle size of the nano hydrogel is in the range of 10nm to 600nm, the viscosity of the nano hydrogel (@ 27 ℃, 1Hz rheometer) is a nano hydrogel for filler treatment in the range of 30Pa · s to 100Pa · s. The method of claim 5,
The nano hydrogel for filler treatment is applied to the surface of the skin without being injected through the subcutaneous, the skin penetration rate of the nano hydrogel is 70 μg / cm ² · 24h or more nano hydrogel for filler treatment. The method of claim 1,
The nano hydrogel for the filler procedure,
Hyaluronic acid aqueous solution having a concentration of 30mg / ml to 40mg / ml, 0.1wt% to 8.0wt% bifunctional crosslinking agent compared to the hyaluronic acid solution and a stem cell culture solution containing HEK293T 0.01wt% to the aqueous hyaluronic acid solution Mix 1.0 wt% of stem cell culture,
Aging the mixture at 45 ° C. to 50 ° C. for at least 24 hours,
A nano hydrogel for filler treatment prepared by reacting the aged mixture at 30 ° C. to 35 ° C. with stirring.

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