KR20210097388A - Hyaluronic acid hydrogel improving viscosity and elasticity - Google Patents

Abstract

Provided is a hyaluronic acid hydrogel with improved viscosity and elasticity. The hydrogel for filler treatment includes the steps of: preparing a first hydrogel by irradiating first electron beams to crosslink an aqueous solution of hyaluronic acid; and forming a second hydrogel by at least partially removing a pendant crosslinked product of the first hydrogel by irradiating second electron beams.

Description

Hyaluronic acid hydrogel with improved viscosity and elasticity {HYALURONIC ACID HYDROGEL IMPROVING VISCOSITY AND ELASTICITY}

According to one aspect of the present invention, the present invention relates to a method for producing a microgel by crosslinking hyaluronic acid, specifically, a microgel for filler treatment that can crosslink hyaluronic acid only by irradiating an electron beam without a crosslinking agent. It relates to a manufacturing method. More specifically, it relates to a microgel for filler treatment, and a method for preparing an injectable filler composition using the same.

According to another aspect of the present invention, the present invention relates to a method for producing a hydrogel by irradiating an electron beam to a polysaccharide, and a hydrogel prepared therefrom. Specifically, it relates to a method for producing a hydrogel in the form of particles by inducing crosslinking without substantially adding a crosslinking agent to a polysaccharide such as hyaluronic acid, and a hydrogel in the form of particles prepared therefrom.

According to another aspect of the present invention, the present invention relates to a method for producing a hydrogel for filler treatment that removes the pendant using an electron beam. In detail, it relates to a method for producing a hydrogel for a filler treatment by crosslinking hyaluronic acid, and relates to a method for producing a hydrogel for a filler treatment that induces removal of a pendant by irradiating an electron beam to the crosslinked hydrogel of hyaluronic acid. .

Hyaluronic acid is one of the complex negatively charged polysaccharides and is a high molecular compound composed of N-acetylglucosamine and glucuronic acid. Hyaluronic acid is mainly present in connective tissue, subcutaneous tissue, nerve tissue, or joint synovial fluid in vivo. Hyaluronic acid is extracted from microbial fermentation of the genus Streptococcus, or the like, and is used in various fields. For example, hyaluronic acid has very good biocompatibility and is used not only as a cosmetic additive, but also as a joint cavity injection, an anti-adhesion agent, and an ophthalmic drug. In addition, hyaluronic acid gel is widely used as a cosmetic filler, which is a tissue restoration biomaterial medical device.

On the other hand, as it has been reported that hyaluronic acid itself can be rapidly excreted from the body by decomposition by enzymes and active oxygen, various attempts have been made to stabilize it by chemically crosslinking it in order to extend the decomposition period in the body.

That is, 1,4-butanediol diglycidyl ether (BDDE), etc., to increase the chemical structural stability of hyaluronic acid and to slow the decomposition in the body to maintain the physical properties for a long period of time. Techniques for stabilizing hyaluronic acid by adding a crosslinking agent of

However, when a crosslinking agent is used to stabilize hyaluronic acid, the unreacted residual crosslinking agent may cause toxicity and cause an inflammatory reaction in the body. There is a problem that not only induces but also weakens the physical properties of the hydrogel.

Kablik J, Monheit GD, Yu LP, Chang G and Gershkovich J. Dermatol Surg 2009, 35(S1): 302-312 (Non-Patent Document 2) Sungchul C, et al. Journal of Biomedical Materials Research Part A, 2014-11-16

Non-Patent Document 1 and Non-Patent Document 2 report that a hydrogel polymer having a pendant cross-linked product lowers physical properties. On the other hand, when an additional cross-linking agent is added to control the physical properties of the hydrogel to a desired level, there is a problem of toxicity that is not suitable for application to the human body. In particular, the FDA requires an unreacted crosslinking agent concentration of 2 ppm or less.

An object of the present invention for solving such a problem is to provide a method for producing a hydrogel for filler treatment that can improve the physical properties of the cross-linked hydrogel and at the same time minimize toxicity.

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

In the method for producing a hydrogel for filler treatment according to an embodiment of the present invention for solving the above problems, a first hydrogel in which at least a portion of the hyaluronic acid is crosslinked by adding a crosslinking agent to an aqueous solution of hyaluronic acid and reacting for a predetermined time manufacturing; sealing the first hydrogel in a first pouch made of a plastic material; Preparing a second hydrogel with increased viscosity and elasticity of the hydrogel by irradiating an electron beam to the first hydrogel sealed in the first pouch; and removing the bubbles of the second hydrogel by opening the first pouch, and repackaging in a second pouch of a material different from that of the first pouch.

In addition, the crosslinking agent may include 1,4-butanediol diglycidyl ether. In some embodiments, the crosslinking agent may consist of only 1,4-butanediol diglycidyl ether.

In addition, the crosslinking agent is 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, 1,2-bis(2,3-epoxypropoxy)ethylene, 1-( 2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,3-butadienediepoxide.

The second hydrogel is formed by at least partially removing a pendant group of the first hydrogel, and the second hydrogel may have a lower pendant crosslinked body formation ratio than that of the first hydrogel.

Here, the formation ratio of the pendant crosslinked body may be calculated according to Equation 1 below.

[Equation 1]

Pendant crosslinked product formation ratio = (pendant crosslinked product amount) / (crosslinked product amount)

Furthermore, the pendant cross-linked body formation ratio of the second hydrogel may be 50% or less of the pendant cross-linked body formation ratio of the first hydrogel.

The step of irradiating the electron beam includes: a) disposing the first hydrogel sealed in the first pouch on a moving table, b) disposing on a moving table including a turntable or a conveyor table fixing the position of the electron beam irradiator so that the electron beam is irradiated to the same position regardless of the change in the position of the first hydrogel, and c) exposing the first hydrogel to the electron beam for a predetermined time by moving the moving table As a step of making the first hydrogel by the moving, it may include a step of exposing the first hydrogel to an electron beam with a predetermined period and a rest period.

Here, the electron beam may be irradiated with an intensity of 0.1 kW to 1.0 kW.

Also, in step c), the period in which the first hydrogel is exposed to the electron beam may be 0.5 seconds to 2 seconds.

Also, in step c), the resting period during which the first hydrogel is not exposed to the electron beam may be 50 seconds or more.

In some embodiments, the period in which the first hydrogel is exposed to the electron beam may be performed a total of 30 or more times.

The first pouch may be made of a polyvinyl chloride film.

In addition, the thickness of the first pouch may be 0.1mm to 0.5mm.

The second pouch may be formed of one selected from the group consisting of a polyethylene film, a polyethylene terephthalate film, and a polypropylene film.

In a preferred embodiment, when the amount of the crosslinking agent added is 0.1 wt% or less compared to the aqueous solution of hyaluronic acid or the reaction time between the aqueous solution of hyaluronic acid and the crosslinking agent is 1 hour or less, the viscosity of the second hydrogel is 400 Pa s or more or elasticity The degree may be 100 Pa or more.

The details of other embodiments are included in the detailed description.

According to embodiments of the present invention, by irradiating an electron-beam to the cross-linked hyaluronic acid hydrogel to induce the removal of pendant or pendant groups, in particular, the reaction of unreacted cross-linking agents such as pendant cross-linking products is induced. can do.

Through this, even if a small amount of the crosslinking agent is used or the reaction time with the crosslinking agent is shortened compared to the conventional method, the physical properties of the hydrogel can be improved to the same or higher level, and the toxicity can also be significantly reduced.

That is, at least a portion of the unreacted cross-linking agent, especially, the pendant cross-linker can induce an additional reaction, so that the physical properties of the hydrogel are improved to the same or higher level even if the cross-linking agent is used in a small amount or the reaction time with the cross-linking agent is shorter than in the conventional method. can do it

In addition, it is possible to reduce the amount of the cross-linking agent used to obtain the desired physical properties, as well as to react and remove the non-reacted cross-linking agent that causes toxicity, such as a pendant cross-linked product, so that the toxicity of the hydrogel can be significantly reduced.

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 chemical formula showing structures that can be formed when BDDE is used as a crosslinking agent for hyaluronic acid.
2 is a chemical formula showing the reaction process of the pendant crosslinked body of BDDE by electron beam irradiation according to an embodiment of the present invention.

Advantages and features of the present invention and methods of achieving them will become 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 will be implemented in various different forms, and only the embodiments allow the disclosure of the present invention to be complete, and those of ordinary skill in the art to which the present invention pertains. It is provided to fully inform the person of the scope of the invention, and the present invention is only defined by the scope of the claims.

That is, various changes may be made to the embodiments presented by the present invention. It should be understood that the embodiments described below are not intended to limit the embodiments, and include all modifications, equivalents or substitutes thereto.

The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. In this specification, 'and/or' includes each and every combination of one or more of the mentioned items. The singular also includes the plural, unless the phrase specifically states otherwise.

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

Hereinafter, the present invention will be described in detail. The first invention, the second invention, and the third invention described below may or may not share some technical ideas with each other. That is, if there is a technical idea that conflicts with each other in the detailed description of the first invention, the second invention, and the third invention, it is applied only to the invention, and technical ideas that do not conflict with each other can be commonly applied to both inventions. It will be readily understood by those of ordinary skill in the art.

First, the first invention will be described in detail.

[First invention]

The method for producing a microgel for filler treatment according to an embodiment of the present invention (ie, an embodiment of the first invention) is 5 kGy in 18w/v% to 25w/v% of hyaluronic acid aqueous solution (first aqueous hyaluronic acid solution). It may include the step of at least partially crosslinking the hyaluronic acid by irradiating an electron beam with a dose of 10 kGy to pulse or impulse. For example, in crosslinking of hyaluronic acid, a crosslinking agent that may cause harm to the human body may be substantially free.

Unless explicitly stated otherwise, the dose unit of the electron beam in the following uses gray (Gy), and may be expressed as energy per weight. In addition, the dose rate unit of the electron beam uses Gy/s, and can be expressed as energy per weight per unit time.

Conventionally, compounds such as 1,4-butanediol diglycidyl ether (BDDE) were added as cross-linking agents to cross-link hyaluronic acid. Since these cross-linking agents are toxic, unreacted cross-linking agents remain and accumulate in the skin, causing inflammation. There was a problem of causing a , and thus, there was a disadvantage that a separate removal process was followed to remove the unreacted crosslinking agent. However, the cross-linking method of hyaluronic acid according to the embodiments of the present invention can cross-link hyaluronic acid only by irradiation with electron beams without a separate cross-linking agent. There is an advantage that a separate process to remove is not required. That is, the microgel according to the present invention is characterized in that it does not contain conventionally used crosslinking agents or glycol-based crosslinking aids.

In another embodiment, the microgel further comprises a crosslinking agent or a crosslinking agent such as polyethylene glycol (poly(ethylene glycol), PEG) and/or polyethylene glycol diglycidyl ether (PEGDE). It may be formed by crosslinking an aqueous solution.

The microgel prepared through electron beam crosslinking is a high-viscosity gel having a viscosity of 15 to 150 Pa·s, preferably about 45 to 150 Pa·s, more preferably about 100 to 150 Pa·s at 1 Hz when measured with a rheometer. As such, it may have an appropriate viscosity for use in filler procedures. In addition, the microgel produced through electron beam crosslinking has an average particle size of about 30 μm to 700 μm, or about 40 μm to 700 μm, or about 50 μm to 700 μm, or about 100 μm to 600 μm, or about 150 μm to 500 μm. μm.

When nano-sized particles are formed because crosslinking of hyaluronic acid does not occur properly, the viscosity is too low to use in Merck gel for filler treatment, so it is important to proceed with crosslinking under thoroughly designed conditions as in the examples of the present invention. do.

If the concentration of hyaluronic acid is lower than 18w/v%, sufficient crosslinking may not be achieved, resulting in a decrease in particle size and a decrease in viscosity. On the other hand, when the concentration of hyaluronic acid exceeds 25w/v%, it is not preferable because aggregation or sticking between particles and further bulk gel may be formed. In this case, the gelation progresses too much, so it is difficult to inject using an extrusion mechanism, etc., and it is not suitable for use in filler procedures.

It is estimated that the dose of the electron beam is closely related to the concentration of the aqueous hyaluronic acid solution. However, as described above, when the present invention uses an aqueous solution of hyaluronic acid in a concentration range of 18w/v% to 25w/v% and irradiates an electron beam with pulse or impulse, the total dose of the electron beam is in the range of 5kGy to 10kGy, or about 5kGy to 9kGy It was confirmed that it is preferable to irradiate in the range of , or in the range of about 5 kGy to 8 kGy. If the dose of the electron beam exceeds 10 kGy, the particle size may be reduced due to the decomposition of hyaluronic acid, and finally the viscosity of the gel may be lowered. On the other hand, when the dose of the electron beam is less than 5 kGy, aggregation or sticking between particles, and further, the formation of bulk gels may occur, which is undesirable.

Those skilled in the art will understand that the dose of the electron beam may be selected in consideration of the weight of the aqueous hyaluronic acid solution.

For example, the energy of the electron beam used in the embodiments of the present invention may be 1 MeV to 100 MeV, but the present invention is not limited thereto. In addition, the hyaluronic acid for preparing the 18w/v% to 20w/v% aqueous solution of hyaluronic acid may use a molecular weight of 100 kDa or more, or about 150 kDa or more, or about 200 kDa or more. If the molecular weight of hyaluronic acid is 100 kDa or less, it is difficult to control the particle size of the microgel, and a particulate hydrogel at the level of several hundred nanometers can be prepared. In addition, the upper limit of the molecular weight of hyaluronic acid may be about 500 kDa, or about 400 kDa. When the molecular weight of hyaluronic acid is too large, the time required for crosslinking is excessive, which may be uneconomical.

In an exemplary embodiment, the irradiation of the electron beam may be performed in a form having a predetermined period. For example, pulse irradiation or impulse irradiation may be used. As used herein, the term 'impulse' means that an impact is applied for an extremely short time, for example, several milliseconds to several tens of milliseconds. In this case, the irradiation period may be 5 to 10 seconds, or about 6 to 8 seconds. If the irradiation cycle is too short, crosslinking may occur like bulk gel, or aggregation or adhesion between particles may occur. If the irradiation cycle is too long, complete crosslinking may not be achieved, crosslinking may occur in a mixed form of colloid and gel, and the viscosity may be significantly reduced.

The total irradiation time during which the electron beam irradiation is performed may be appropriately adjusted depending on the shape of the microgel to be crosslinked, but may be, for example, about 10 to 30 minutes. If the total irradiation time is too short, sufficient crosslinking may not be achieved, and if the total irradiation time is too long, the particle size of the hydrogel in the form of particles may become non-uniform. In addition, if the irradiation time is too long, the turbidity of the hydrogel may increase.

In the case of irradiating the electron beam with a pulse, the unit irradiation time for which the electron beam is irradiated within one cycle, that is, the pulse width may be about 3 seconds or less, or about 2.5 seconds or less, preferably about 2 seconds or less. When the pulse width exceeds 3 seconds under the conditions of concentration and dose as described above, aggregation or adhesion between particles, furthermore, a phenomenon in which a gel in a bulk form is formed may occur. The lower limit of the pulse width is not particularly limited, but may be, for example, 1 millisecond or more, or about 10 milliseconds or more, or about 100 milliseconds or more.

In some non-limiting embodiments, when the electron beam is irradiated with pulses, it may be desirable to gradually increase the dose of the electron beam. For example, as the number of irradiation of the electron beam increases, the dose rate may gradually increase. That is, despite the change in the dose rate, the total dose may all be within the range of 5 kGy to 10 kGy. Although the present invention is not limited to any theory, it is assumed that the crosslinking efficiency gradually decreases in the process of microgelation due to electron beam irradiation, so it may be desirable to gradually increase the dose rate (the amount of energy per unit time) .

In some embodiments, the step of crosslinking hyaluronic acid by irradiating electron beams may be performed at a temperature of 50°C to 60°C.

On the other hand, the first aqueous solution of hyaluronic acid may further include a derivative of genipin. The genipin derivative may have an epoxy group as represented by the following formula (1).

[Formula 1]

Genipine and derivatives of genipin may easily react with hyaluronic acid and may be cross-linked with hyaluronic acid. That is, genipine and genipine derivatives may function as crosslinking agents (biocompatible crosslinking agents), but the functions of genipine and genipine derivatives are not limited to the term 'crosslinking agent'. Genipine has very low biotoxicity (LD50 i.v. 382 mg/kg in mice), so the removal of residual components is practically unnecessary, while the physical properties of the manufactured Merck gel can be improved. That is, the crosslinking rate of the hydrogel can be increased, and the genipine derivative can form an intermolecular crosslink with hyaluronic acid. On the other hand, although the present invention is not limited thereto, cross-linking between molecules of the genipine derivative may not be substantially achieved.

The inventors of the present invention have found that crosslinking efficiency is increased when a derivative of genipin, not genipin, is mixed in the preparation of microgels by irradiating electron beams to hyaluronic acid, and thus has completed the present invention. That is, the present invention is not limited thereto, but in the best mode of the present invention, the aqueous hyaluronic acid solution may substantially contain no genipin.

The content range of the genipine derivative may be about 0.1 wt% to 0.5 wt% compared to hyaluronic acid. If the content of the genipine derivative exceeds the above range, the microgel is not transparent and becomes opaque, so it may be difficult to apply as a filler composition.

Meanwhile, in some embodiments, after preparing the first aqueous hyaluronic acid solution as described above, and starting to irradiate an electron beam to the first aqueous solution of hyaluronic acid in a pulse or impulse form, the first aqueous solution of hyaluronic acid and the second aqueous solution of hyaluronic acid are It may further include the step of mixing. The step of mixing the second aqueous solution of hyaluronic acid may be performed simultaneously with the irradiation of the electron beam. That is, the second aqueous solution of hyaluronic acid may be further mixed while irradiating the electron beam, and the irradiation of the electron beam may be continuously performed without stopping.

The second aqueous hyaluronic acid solution may have a higher concentration than the first aqueous hyaluronic acid solution. For example, the lower limit of the concentration of the second aqueous solution of hyaluronic acid is about 20 w/v%, or about 21 w/v%, or about 22 w/v%, or about 23 w/v%, or about 24 w/v%, or about 25w/v%.

Both hyaluronic acid in the first aqueous solution of hyaluronic acid and hyaluronic acid in the second aqueous solution of hyaluronic acid may contribute to the formation of microgels. When additional hyaluronic acid is mixed and crosslinked according to the present embodiment, the particle size uniformity of the microgel can be improved and elasticity can be further improved. If the concentration of the second aqueous solution of hyaluronic acid is smaller than the concentration of the first aqueous solution of hyaluronic acid, crosslinking between the first and second hyaluronic acid is not made and the particle size distribution may be very large. That is, the second hyaluronic acid is not preferable because it can form a nano-sized gel rather than a microgel.

On the other hand, the upper limit of the concentration of the second aqueous solution of hyaluronic acid may be about 30w / v%. When the concentration of the second aqueous hyaluronic acid solution exceeds 30w/v%, a bulk gel may be prepared and the particle size may be excessively large. The molecular weight of the second hyaluronic acid may be the same as or different from that of the first hyaluronic acid.

In some embodiments, mixing between the first hyaluronic acid and the second hyaluronic acid may be performed through stirring. In this case, a homogenizer or the like may be used for the stirring, but the present invention is not limited thereto. The lower limit of the stirring speed is not particularly limited, but may be about 500 rpm or more in terms of efficient stirring. On the other hand, if the stirring speed is too large, the formed microgel may be pulverized or effective crosslinking may not be achieved. The upper limit of the stirring speed may be about 1,000 rpm.

As described above, when a hydrogel having a micro size, that is, a microgel, is used for a filler procedure, the particle size uniformity of the particles is a very important factor. According to the present invention, it is possible to obtain a microgel having a particle size of several tens of micrometers to several hundred micrometers and having excellent particle size uniformity.

The hydrogel prepared according to the above-described embodiments of the present invention may be additionally subjected to a known addition process or sterilization process to be used as a Merck gel for filler treatment.

For example, the hydrogel for filler treatment of the present invention may include a generally physiologically acceptable carrier fluid, such as an isotonic buffer, particularly preferably a buffered physiological saline solution.

In an exemplary embodiment, the hydrogel for filler treatment of the present invention may contain 1 to 20 parts by weight of the composition for improving the persistence of hyaluronic acid in the body compared to 100 parts by weight of the composition.

The hydrogel for filler treatment of the present invention is preferably used to treat cosmetic diseases, such as wrinkles or wrinkles of the skin (eg, facial wrinkles and facial wrinkles), glabellar lines, nasolabial folds, chin wrinkles, marionette wrinkles , oral cavity, periorbital folds, fine lines around the eyes, skin depressions, scars, temples, subdermal supports of eyebrows, cheek and cheek fat pads, lacrimal gullies, nose, lips, cheeks, periorbital area, suborbital area, facial asymmetry , for the treatment of the lower jaw line and the jaw. Alternatively, it may be administered to treat therapeutic indications, such as stress incontinence, bladder-ureteral reflux, vocal fold insufficiency, vocal fold mediation.

Hereinafter, embodiments of the present invention will be described in more detail through specific preparation examples and experimental examples. However, the following preparation examples and experimental examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1: Preparation of Merck hydrogel (Examples 1-1 to 3-3)

About 200 kDa hyaluronic acid is dissolved in water to prepare 5, 15 and 30 w/v% aqueous solutions, and electron beams are applied to the prepared hyaluronic acid aqueous solution for 1 minute using a direct current electron-beam accelerator. Hydrogels of Examples 1-1 to 3-3 were prepared by inducing crosslinking of hyaluronic acid by irradiating it at a dose as shown in Table 1 below.

Dose/concentration 5 w/v% 15 w/v% 30 w/v% 0.1 kGy Example 1-1 Example 2-1 Example 3-1 3 kGy Example 1-2 Example 2-2 Example 3-2 10 kGy Examples 1-3 Example 2-3 Example 3-3

Experimental Example 1: Measurement of the viscosity (viscosity) of Merck hydrogel

Viscosity at 1 Hz of Examples 1-1 to 3-3 was measured with an Anton-Paar MCR 302 rheometer, and the results are shown in Table 2 below.

Viscosity (Pa s) Example 1-1 0.038 Example 2-1 0.051 Example 3-1 0.17 Example 1-2 196 Example 2-2 882 Example 3-2 1050 Examples 1-3 2 Example 2-3 101 Example 3-3 244

Viscosity as shown in Table 2 may indicate the degree of bulking or gelation of Examples 1-1 to 3-3. In the case of Examples 1-1, 1-3, 2-1, and 3-1, the viscosity was very low and crosslinking was not sufficiently achieved, or it was assumed that nano-sized particles were formed. In addition, in the case of Examples 2-3 and 3-3, it was confirmed that Merck Gel did not have a uniform viscosity but had partially different viscosities, apart from the fact that bulking was made to a considerable degree. In particular, in the case of Example 3-3, the degree of partial opacity was severe.

As described above, it can be seen that the closer the dose of the electron beam is to 3 kGy, the higher the concentration of hyaluronic acid is, the better the crosslinking is, and the Merck gel having high viscosity and viscoelasticity can be obtained. This is presumed to be because if the dose of the electron beam is too low than 3 kGy, the generation of free radicals is difficult, so that cross-linking does not occur sufficiently, and if it is too high, the hyaluronic acid chain is cut and decomposed. However, it goes without saying that the present invention is not limited to any theory.

Preparation Example 2: Preparation of microgels (Example 4)

This time, the concentration of hyaluronic acid and the dose of electron beam, which were confirmed to be the most preferable through Preparation Example 1, were irradiated with an electron beam of 3 kGy to a 15w/v% aqueous solution of about 200 kDa hyaluronic acid, and the electron beam was pulsed by irradiating the hyaluronic acid. The hydrogel of Example 4 was prepared by inducing crosslinking.

Specifically, the pulse irradiation of the electron beam was made to have a period of 7 seconds for a total of 20 minutes, and within each period, the electron beam was irradiated for 2 seconds and had a rest period of 5 seconds.

Experimental Example 2: Confirmation of particle size and viscosity of microgel

The particle size of the hydrogel of Example 4 was confirmed by DLS (Dynamic light scattering), and the result was as shown in FIG. 1 . In addition, the viscosity measured at 1 Hz of Example 4 with an Anton-Paar MCR 302 rheometer was 37 Pa·s.

As shown in FIG. 1 , it can be seen that a microgel having a particle size suitable for immediate use for a filler procedure was prepared unlike the Merck gel prepared in Preparation Example 1 by pulse irradiation with an electron beam.

Preparation Example 3: Preparation of microgels (Examples 5-1 to 5-6)

Examples 5-1 to 5-6 by inducing crosslinking of hyaluronic acid by pulsed irradiation of electron beams in the same manner as in Preparation Example 2, but by varying the concentration of the aqueous solution of hyaluronic acid and the total dose of electron beams as shown in Table 3 below. of the hydrogel was prepared.

Concentration/dose 5 kGy 10 kGy 18w/v% Example 5-1 Example 5-4 22w/v% Example 5-2 Example 5-5 25w/v% Example 5-3 Example 5-6

Experimental Example 3: Confirmation of particle size of microgel

The particle size of the hydrogels of Examples 5-1 to 5-6 was confirmed by DLS.

2 is a result of confirming the particle size of the hydrogel of Examples 5-1, 5-2 and 5-6 with DLS among them, (A) to (C) are Examples 5-1, 5-2 and 5-6. As shown in FIG. 2, the hydrogel particles of Examples 5-1, 5-2 and 5-6 are more uniform than the hydrogel particles of Example 4 shown in FIG. It can be seen that the particles are uniform.

Preparation Example 4: Preparation of microgels (Examples 6-1 to 6-3)

Examples 6-1 to 6-3 by inducing crosslinking of hyaluronic acid by using the same hyaluronic acid concentration and electron beam dose as in Example 5-2 of Preparation Example 3, but using a different pulse method as shown in Table 4 below. of the hydrogel was prepared.

Irradiation time within each cycle
(unit irradiation time) total investigation time Example 6-1 4 seconds 20 minutes Example 6-2 2 seconds 5 minutes Example 6-3 2 seconds 80 minutes

Experimental Example 4: Confirmation of particle size of microgel

The particle size of the hydrogels of Examples 6-1 to 6-3 was confirmed by DLS.

3 is a result of confirming the particle size of the hydrogel by DLS, (A) to (C) show Examples 6-1, 6-2 and 6-3, respectively. As shown in FIG. 3 , it can be seen that the particle size distribution becomes non-uniform when the unit irradiation time, which is the irradiation time within each cycle, is longer than 2 seconds or the total irradiation time is too short or longer than 20 minutes. In addition, in the case of Examples 6-2 and 6-3, it was confirmed that the particle size was excessively reduced.

Preparation Example 5: Mixing a high-concentration aqueous hyaluronic acid solution to prepare microgels (Examples 7-1 to 7-3)

In the same manner as in Example 5-2 of Preparation Example 3, while irradiating an electron beam to the aqueous solution of hyaluronic acid in a pulse, at the same time, another aqueous solution of about 200 kDa of hyaluronic acid was added little by little to the aqueous solution of hyaluronic acid and mixed with a stirrer. Hydrogels of Examples 7-1 to 7-3 were prepared by inducing crosslinking of hyaluronic acid by varying the stirring speed of the stirrer and the concentration of the added hyaluronic acid aqueous solution as shown in Table 5 below.

Stirring speed (rpm) Concentration (w/v%) Example 7-1 1000 27 Example 7-2 2000 27 Example 7-3 1000 15

Experimental Example 5: Confirmation of particle size of microgel

The particle size of the hydrogels of Examples 7-1 and 7-2 was confirmed by DLS.

4 is a result of confirming the particle size of the hydrogel by DLS, (A) and (B) show Examples 7-1 and 7-2, respectively.

As shown in FIG. 4, when a relatively high concentration of hyaluronic acid aqueous solution is mixed with pulse irradiation of an electron beam, a microgel having a uniform particle size can be obtained and the particle size can be increased than in Example 5-2, but It can be seen that, as in Example 7-2, if the stirring speed is too high (2000 rpm) when mixing, the particles are pulverized, which is undesirable.

On the other hand, in Example 7-3, it was difficult to obtain a clear peak on the DLS, and it can be inferred that the particle size distribution becomes very non-uniform when the concentration of the added hyaluronic acid aqueous solution is relatively low.

Preparation Example 6: Preparation of microgels by adding a biocompatible crosslinking agent (Examples 8-1 and 8-2)

In the same manner as in Example 5-2 of Preparation Example 3, an electron beam is irradiated to the aqueous solution of hyaluronic acid by pulse, wherein the aqueous solution of hyaluronic acid is a biocompatible crosslinking agent Genipin (Sigma-Aldrich) and the formula 1 Those in which the derivative was added in an amount of 0.5 wt% relative to hyaluronic acid, respectively (Examples 8-1 and 8-2) were used. The genipine derivative of Formula 1 below was prepared using a known synthesis method. (J. Luo, et al., ChemMedChem (2012) 1661-8)

[Formula 1]

Experimental Example 6: Confirmation of the viscosity of microgels

Viscosity at 1 Hz of Examples 7-1 and 7-2 was measured with an Anton-Paar MCR 302 rheometer, and the results are shown in Table 6 below.

Viscosity (Pa s) Example 7-1 45 Example 7-2 101

As shown in Table 6, when genipine was added, there was no significant change in the viscosity of the microgel, whereas when the genipine derivative represented by Formula 1 was added, the viscosity of the microgel was significantly improved. can

As described above, it has been reported that nano-sized particles are formed by irradiating electron beams on biocompatible polymers such as hyaluronic acid. However, nano-sized particles have a limit that cannot be applied as a hydrogel for filler treatment, and the inventor of the present invention has completed the present invention by attempting to form a micro-sized gel.

According to the present invention, it is possible to form microgels only by electron beam crosslinking, rather than simply increasing the particle size from nano size to micro size. In addition, since the microgel has an excellent particle size and viscosity, there is an effect that it can be directly applied as a hydrogel for a filler procedure and the like.

Hereinafter, the second invention will be described in detail.

[Second invention]

(A) Method for producing hydrogel

The method for producing a hydrogel according to an embodiment of the present invention (ie, an embodiment of the second invention) comprises the steps of (a) preparing an aqueous polysaccharide solution, and (b) irradiating electron beams to form hydrogel particles may include. Hereinafter, each step will be described.

(a) preparing a polysaccharide aqueous solution

As used herein, the term 'polysaccharide' refers to a molecule in which three or more monosaccharides form a glycosidic bond. Polysaccharide may be used in the sense of including oligosaccharides. The glycosidic linkage may include α-glycosides and β-glycosides. The polysaccharide may have excellent reactivity because it has a hydroxyl group, an amino group, and/or a carboxylic acid group in the molecule.

The polysaccharide preferably has biocompatibility and water solubility. Examples of polysaccharides that can be used in the present invention include gelatin, chitosan, collagen, mannan, dextran, dextran sulfate, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, maltodextrin. ), fructooligosaccharide, isomaltooligosaccharide, inulin, hyaluronic acid, alginate, glycogen, amylopectin, amylose, sucrose, agarose, galactose, carboxymethyldextran, beta-glucan, epichlorohydrin , hydroxyethyl cellulose, carboxymethyl cellulose, fucoidan (fucoidan), may be at least one selected from the group consisting of chondroitin and derivatives thereof. Preferably, it may be chitosan, mannan, hyaluronic acid, cyclodextrin, dextran, alginate, fructooligosaccharide, isomaltooligosaccharide, fucoidan or carboxymethyldextran, or a derivative thereof. More preferably, it may be hyaluronic acid, dextran, or fucoidan, or a derivative thereof.

These polysaccharides can form hydrogels by intermolecular or intramolecular crosslinking. The crosslinking of polysaccharides may be performed by irradiation with electron beams, as will be described later, and may proceed without mixing of an additional crosslinking agent. That is, the cross-linking agent may increase the viscoelasticity of the cross-linked product, but the residual cross-linking agent that is decomposed or unreacted in vivo may cause toxicity and cause an inflammatory reaction in the body. Therefore, in order to remove the unreacted crosslinking agent, a crosslinking agent removal process, etc. must be performed, which may cause uneconomical efficiency. On the other hand, according to the manufacturing method according to the present invention, there is an advantage that the polysaccharide can be cross-linked only by irradiation with an electron beam.

The concentration of the polysaccharide aqueous solution may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the concentration of the aqueous polysaccharide solution is about 0.1% (w/v), or about 0.5% (w/v), or about 0.7% (w/v), or about 1.0% (w/v), or about 2.0% (w/v), or about 3.0% (w/v), or about 4.0% (w/v), or about 5.0% (w/v), or about 6.0% (w/v), or about 7.0% (w/v), or about 8.0% (w/v), or about 9.0% (w/v), or about 10.0% (w/v), or about 11.0% (w/v), or about 12.0% (w/v), or about 13.0% (w/v), or about 14.0% (w/v), or about 15.0% (w/v), or about 16.0% (w/v), or about 17.0% (w/v), or about 18.0% (w/v), or about 19.0% (w/v), or about 20.0% (w/v), or about 21.0% (w/v), or about 22.0% (w/v), or about 23.0% (w/v), or about 24.0% (w/v), or about 25.0% (w/v), or about 30.0% (w/v), or about 35.0% (w/v), or about 40.0% (w/v), or about 40.0% (w/v), or about 50.0% (w/v).

When a plurality of numerical values are described herein, it can be understood that the specification discloses numerical ranges having any two numerical values as upper and lower limits, respectively. For example, the concentration of the polysaccharide aqueous solution is about 0.1% (w / v) to 10.0% (w / v), about 1.0% (w / v) to 15.0% (w / v), or about 5.0% (w) /v) to 20.0% (w/v). Also, when two or more numerical values are described herein, it can be understood that the present specification also discloses any numerical value present between the two numerical values. For example, the concentration of the polysaccharide aqueous solution may be about 0.8% (w/v) or about 0.9% (w/v).

The concentration of polysaccharide in the polysaccharide aqueous solution may affect the shape of the hydrogel formed by irradiation with electron beams. For example, when the concentration of the aqueous solution is too high, a viscoelastic body having a high degree of crosslinking and viscosity may be formed, and when the concentration of the aqueous solution is too low, a viscoelastic body having a low degree of crosslinking and viscosity may be formed.

The molecular weight of the polysaccharide or its derivative may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the molecular weight of the polysaccharide is about 0.1 kDa, or about 0.5 kDa, or about 0.6 kDa, or about 0.7 kDa, or about 0.8 kDa, or about 0.9 kDa, or about 1.0 kDa, or about 1.1 kDa, or about 1.2 kDa, or about 1.3 kDa, or about 1.4 kDa, or about 1.5 kDa, or about 1.6 kDa, or about 1.7 kDa, or about 1.8 kDa, or about 1.9 kDa, or about 2.0 kDa, or about 3.0 kDa, or about 4.0 kDa, or about 5.0 kDa, or about 6.0 kDa, or about 7.0 kDa, or about 8.0 kDa, or about 9.0 kDa, or about 10 kDa, or about 11 kDa, or about 12 kDa, or about 13 kDa, or about 14 kDa, or about 15 kDa , or about 16 kDa, or about 17 kDa, or about 18 kDa, or about 19 kDa, or about 20 kDa, or about 30 kDa, or about 40 kDa, or about 50 kDa, or about 60 kDa, or about 70 kDa, or about 80 kDa, or about 90 kDa, or about 100 kDa, or about 150 kDa, or about 200 kDa, or about 250 kDa, or about 300 kDa, or about 350 kDa, or about 400 kDa, or about 450 kDa, or about 500 kDa, or about 600 kDa, or about 700 kDa, or about 800 kDa, or about 900 kDa , or about 1,000 kDa, or about 1,200 kDa, or about 1,400 kDa, or about 1,600 kDa, or about 1,800 kDa, or about 2,000 kDa, or about 2,500 kDa, or about 3,000 kDa, or about 3,500 kDa, or about 4,000 kDa , or about 4,500 kDa, or about 5,000 kDa. As described above, it can be understood that the molecular weight of the polysaccharide according to the present invention discloses a numerical range having any two values described above as an upper limit and a lower limit, respectively, and/or a numerical value between any two numerical values. .

The pH of the polysaccharide aqueous solution may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the pH of the aqueous polysaccharide solution is about 0.5, or about 1.0, or about 1.5, or about 2.0, or about 2.5, or about 3.0, or about 3.5, or about 4.0, or about 4.5, or about 5.0, or about 5.5 , or about 6.0, or about 6.5, or about 7.0, or about 7.5, or about 8.0, or about 8.5, or about 9.0, or about 9.5, or about 10.0, or about 10.5, or about 11.0, or about 11.5, or about 12.0, or about 12.5, or about 13.0, or about 13.5, or about 14.0. As described above, the pH of the aqueous polysaccharide solution according to the present invention may be understood to disclose a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values. there is.

The viscosity of the polysaccharide aqueous solution may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the viscosity of the aqueous polysaccharide solution may be 0.005 Pa·s, or about 0.01 Pa·s, or about 0.02 Pa·s, or about 0.03 Pa·s, or about 0.04 Pa·s, or about 0.05 Pa·s, or about 0.06 Pa·s, or about 0.07 Pa·s, or about 0.08 Pa·s, or about 0.09 Pa·s, or about 0.1 Pa·s, or about 0.2 Pa·s, or about 0.3 Pa·s, or about 0.4 Pa·s, or about 0.5 Pa·s, or about 0.6 Pa·s, or about 0.7 Pa·s, or about 0.8 Pa·s, or about 0.9 Pa·s, or about 1.0 Pa·s. As described above, the viscosity of the polysaccharide aqueous solution according to the present invention can be understood as disclosing a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values. there is.

In some embodiments, the polysaccharide aqueous solution may further include a glycol material such as polyethylene glycol (poly(ethylene glycol), PEG) and/or polyethylene glycol diglycidyl ether (PEGDE). .

The concentration of polyethylene glycol alone, polyethylene glycol diglycidyl ether alone, or a mixture thereof in the polysaccharide aqueous solution is about 0.1% (w/v), or about 0.5% (w/v), or about 0.7% (w/v) ), or about 1.0% (w/v), or about 2.0% (w/v), or about 3.0% (w/v), or about 4.0% (w/v), or about 5.0% (w/v) ), or about 6.0% (w/v), or about 7.0% (w/v), or about 8.0% (w/v), or about 9.0% (w/v), or about 10.0% (w/v) ), or about 11.0% (w/v), or about 12.0% (w/v), or about 13.0% (w/v), or about 14.0% (w/v), or about 15.0% (w/v) ), or about 16.0% (w/v), or about 17.0% (w/v), or about 18.0% (w/v), or about 19.0% (w/v), or about 20.0% (w/v) ), or about 21.0% (w/v), or about 22.0% (w/v), or about 23.0% (w/v), or about 24.0% (w/v), or about 25.0% (w/v) ), or about 30.0% (w/v), or about 35.0% (w/v), or about 40.0% (w/v), or about 40.0% (w/v), or about 50.0% (w/v) ) can be As described above, it can be understood that the concentration of glycol according to the present invention discloses a numerical range having any two numerical values as upper and lower limits, respectively, and/or a numerical value between any two numerical values described above. .

In addition, in order to promote crosslinking, an accelerator such as carbon tetrachloride may be further included. In addition, a viscosity modifier may be further included to adjust the viscosity. In addition, the polysaccharide aqueous solution may not contain a crosslinking agent such as 1,4-butanediol diglycidyl ether (BDDE) which is conventionally used. Furthermore, the polysaccharide aqueous solution may not contain an organic solvent.

(b) irradiating the electron beam

This step may be a step of inducing crosslinking of the aforementioned polysaccharides. That is, polysaccharides can form hydrogels by intermolecular or intramolecular crosslinking. For example, covalent bonds present in polysaccharides may be broken and radicals containing lone pairs of electrons may be formed. Accordingly, crosslinking may occur, but of course, the present invention is not limited to any theory.

The irradiation amount of the electron beam, that is, the dose may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the dose of the electron beam is about 0.01 kGy, or about 0.05 kGy, or about 0.1 kGy, or about 0.2 kGy, or about 0.3 kGy, or about 0.4 kGy, or about 0.5 kGy, or about 1.0 kGy, or about 1.5 kGy. , or about 2.0 kGy, or about 2.5 kGy, or about 3.0 kGy, or about 3.5 kGy, or about 4.0 kGy, or about 4.5 kGy, or about 5.0 kGy, or about 5.5 kGy, or about 6.0 kGy, or about 6.5 kGy , or about 7.0 kGy, or about 7.5 kGy, or about 8.0 kGy, or about 8.5 kGy, or about 9.0 kGy, or about 9.5 kGy, or about 10.0 kGy, or about 11.0 kGy, or about 12.0 kGy, or about 13.0 kGy , or about 14.0 kGy, or about 15.0 kGy, or about 20.0 kGy, or about 25.0 kGy, or about 30.0 kGy, or about 40.0 kGy, or about 50.0 kGy, or about 60.0 kGy, or about 70.0 kGy, or about 80.0 kGy , or about 90.0 kGy, or about 100.0 kGy, or about 150.0 kGy, or about 200.0 kGy, or about 250.0 kGy, or about 300.0 kGy, or about 350.0 kGy, or about 400.0 kGy, or about 450.0 kGy, or about 500.0 kGy can be As described above, it can be understood that the dose of electron beam according to the present invention discloses a numerical range having any two values described above as an upper limit and a lower limit, respectively, and/or a numerical value between any two numerical values. .

The irradiation time of the electron beam may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the irradiation time of the electron beam is about 1.0 second, or about 2.0 seconds, or about 3.0 seconds, or about 4.0 seconds, or about 5.0 seconds, or about 10.0 seconds, or about 15.0 seconds, or about 20.0 seconds, or about 25.0 seconds. seconds, or about 30.0 seconds, or about 35.0 seconds, or about 40.0 seconds, or about 45.0 seconds, or about 50.0 seconds, or about 55.0 seconds, or about 60.0 seconds, or about 90.0 seconds, or about 120.0 seconds, or about 150.0 seconds seconds, or about 180.0 seconds, or about 210.0 seconds, or about 240.0 seconds, or about 270.0 seconds, or about 300.0 seconds, or about 360.0 seconds, or about 420.0 seconds, or about 480.0 seconds, or about 540.0 seconds, or about 600 seconds, or about 11 minutes, or about 12 minutes, or about 13 minutes, or about 14 minutes, or about 15 minutes, or about 16 minutes, or about 17 minutes, or about 18 minutes, or about 19 minutes, or about 20 minutes, about 30 minutes, or about 40 minutes, or about 50 minutes, or about 60 minutes, or about 70 minutes, or about 80 minutes, or about 90 minutes, or about 100 minutes, or about 110 minutes, or about 120 minutes. , or about 150 minutes, or about 180 minutes, or about minutes, or about minutes, or about minutes, or about 300 minutes, or about 6 hours, or about 7 hours, or about 8 hours, or about 9 hours, or about It can be 10 hours. As described above, it can be understood that the irradiation time of the electron beam according to the present invention discloses a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values. there is.

In some embodiments, in the irradiation of the electron beam for crosslinking of the polysaccharide, the irradiation of the electron beam may be performed with a resting period in the middle. As a non-limiting example, after irradiating the electron beam for about 10 seconds, without irradiating the electron beam for about 20 seconds, it is possible to start the irradiation of the electron beam again. This process may be performed multiple times.

The dose rate of the electron beam can be understood as the total dose divided by the irradiation time. The dose rate of the electron beam may be appropriately selected in consideration of the dose and irradiation time. In some embodiments, in the irradiation of electron beams for crosslinking polysaccharides, the dose rate of electron beams may be irradiated with a pattern such as increasing, decreasing, or maintaining. As a non-limiting example, while increasing the dose rate of the electron beam for a predetermined time, maintaining the dose rate of the electron beam for a predetermined time, it is possible to decrease the dose rate of the electron beam for a predetermined time again. This process may be performed multiple times.

While the electron beam is irradiated, the polysaccharide aqueous solution may be maintained at a predetermined temperature. The temperature may be appropriately selected in consideration of the type of polysaccharide and crosslinking reactivity. For example, the crosslinking temperature may be about 15°C, or about 20°C, or about 25°C, or about 30°C, or about 31°C, or about 32°C, or about 33°C, or about 34°C, or about 35°C, or about 36°C, or about 37°C, or about 38°C, or about 39°C, or about 40°C, or about 41°C, or about 42°C, or about 43°C, or about 44°C, or about 45°C, or about 46°C, or about 47°C, or about 48°C, or about 49°C, or about 50°C, or about 51°C, or about 52°C, or about 53°C, or about 54°C, or about 55°C, or about 60°C, or about 65°C, or about 70°C, or about 75°C, or about 80°C. As described above, the crosslinking temperature according to the present invention can be understood as disclosing a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values.

In some embodiments, in the irradiation of the electron beam for crosslinking of the polysaccharide, the temperature may have a pattern such as increasing, decreasing, or maintaining. As a non-limiting example, the temperature may be increased for a predetermined time, the temperature may be maintained for a predetermined time, and then the temperature may be decreased again for a predetermined time. This process may be performed multiple times.

While the electron beam is irradiated, the polysaccharide aqueous solution may be stirred using a homogenizer or the like. For example, the stirring speed may be about 500 rpm, or about 1,000 rpm, or about 1,100 rpm, or about 1,200 rpm, or about 1,300 rpm, or about 1,400 rpm, or about 1,500 rpm, or about 1,600 rpm, or about 1,700 rpm, or about 1,800 rpm, or about 1,900 rpm, or about 2,000 rpm, or about 2,100 rpm, or about 2,200 rpm, or about 2,300 rpm, or about 2,400 rpm, or about 2,500 rpm, about 2,600 rpm, or about 2,700 rpm, or about 2,800 rpm, or about 2,900 rpm, or about 3,000 rpm, or about 3,500 rpm, or about 4,000 rpm, or about 4,500 rpm, or about 5,000 rpm, or about 6,000 rpm. As described above, the stirring speed according to the present invention can be understood as disclosing a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values.

(c) encapsulating the loading body

In some embodiments, a loading body or the like may be mounted on the cross-linked hydrogel. The loading process may be performed by adding a loading body to the particulate hydrogel and then stirring. The stirring speed is not particularly limited, but the stirring speed is 100 rpm, or about 200 rpm, or about 300 rpm, or about 400 rpm, or about 500 rpm, or about 1,000 rpm, or about 1,100 rpm, or about 1,200 rpm, or about 1,300 rpm, or about 1,400 rpm, or about 1,500 rpm, or about 1,600 rpm, or about 1,700 rpm, or about 1,800 rpm, or about 1,900 rpm, or about 2,000 rpm, or about 2,100 rpm, or about 2,200 rpm, or about 2,300 rpm, or about 2,400 rpm, or about 2,500 rpm, about 2,600 rpm, or about 2,700 rpm, or about 2,800 rpm, or about 2,900 rpm, or about 3,000 rpm, or about 3,500 rpm, or about 4,000 rpm, or about 4,500 rpm, or about 5,000 rpm, or about 6,000 rpm. As described above, the stirring speed according to the present invention can be understood as disclosing a numerical range in which any two numerical values described above are respectively an upper limit and a lower limit, and/or a numerical value between any two numerical values.

The loading body may include a known one commonly used in drug delivery systems.

For example, the loading body may include a drug. In addition, drugs include antibiotics, anticancer drugs, analgesics, anti-inflammatory drugs, antitussives, expectorants, sedatives, muscle relaxants, epilepsy drugs, ulcer drugs, antidepressants, anti-allergic drugs, cardiac drugs, antiarrhythmic drugs, vasodilators, diuretics, diabetes drugs, anticoagulants, It may be one or more selected from the group consisting of a hemostatic agent, an anti-nodulatory agent, a hormonal agent, and combinations thereof.

For another example, the loading body may include a diagnostic marker. Examples of diagnostic markers include fluorescent substances, radioactive isotopes, contrast agents, and the like. Specifically, the radiolabeled material for computed tomography (CT) may include ^99m Tc, ¹²³ I, ¹²⁵ I, ¹¹¹ In, ⁶⁷ Ga, ¹⁷⁷ Lu, ²⁰¹ Tl, and ^117m Sn. In addition, the labeling material for positron tomography may include ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ³⁸ K, ⁶² Cu, ⁶⁴ Cu, ⁶⁸ Ga, ⁸² Rb, ¹²⁴ I and ⁸⁹ Zr. In addition, examples of the fluorescent material include quantum dots such as CdSe, CdS, ZnS, and ZnSe.

Examples of the ultrasound contrast agent include perfluoropropane, perfluorohexane, sulfurhexafluoride, perfluoropentane and decafluorobutane.

(B) hydrogel

The hydrogel prepared according to the present invention may have a particle form. That is, the hydrogel according to the present invention may have a nano size or a micro size. For example, the average particle size of the hydrogel is about 10 nm, or about 20 nm, or about 30 nm, or about 40 nm, or about 50 nm, or about 60 nm, or about 70 nm, or about 80 nm, or about 90 nm, or about 100 nm, or about 150 nm, or about 200 nm, or about 250 nm, or about 300 nm, or about 350 nm, or about 400 nm, or about 450 nm, or about 500 nm, or about 550 nm, or about 600 nm, or about 700 nm, or about 800 nm, or about 900 nm can be or the average particle size of the hydrogel is about 10 μm, or about 20 μm, or about 30 μm, or about 40 μm, or about 50 μm, or about 60 μm, or about 70 μm, or about 80 μm, or about 90 μm , or about 100 μm, or about 150 μm, or about 200 μm, or about 250 μm, or about 300 μm, or about 350 μm, or about 400 μm, or about 450 μm, or about 500 μm, or about 550 μm , or about 600 μm, or about 650 μm, or about 700 μm, or about 750 μm, or about 800 μm. As described above, the average particle size of the hydrogel according to the present invention is to be understood as disclosing a numerical range having any two values described above as an upper limit and a lower limit, respectively, and/or a numerical value between any two numerical values. can

(C) hydrogel composition

The particulate hydrogel according to the invention may generally comprise a physiologically acceptable carrier fluid, such as an isotonic buffer, particularly preferably a buffered physiological saline solution.

In some embodiments, the hydrogel composition may further include a lubricant, a wetting agent, an emulsifying agent, a suspending agent, a preservative, a pH adjusting agent and/or a viscosity adjusting agent, and the like.

The viscosity of the hydrogel composition may be measured at 20° C. and 1 Hz with a rheometer. For example, the viscosity of the hydrogel on the particle may be about 0.0001 Pa.s, or about 0.001 Pa.s, or about 0.01 Pa.s, or about 0.1 Pa.s, or about 1.0 Pa.s, or about 2.0 Pa.s. s, or about 3.0 Pa-s, or about 4.0 Pa-s, or about 5.0 Pa-s, or about 6.0 Pa-s, or about 7.0 Pa-s, or about 8.0 Pa-s, or about 9.0 Pa-s , or about 10.0 Pa s, or about 11.0 Pa s, or about 12.0 Pa s, or about 13.0 Pa s, or about 14.0 Pa s, or about 15.0 Pa s, or about 16.0 Pa s, or about 17.0 Pa-s, or about 18.0 Pa-s, or about 19.0 Pa-s, or about 20.0 Pa-s, or about 25.0 Pa-s, or about 30.0 Pa-s, or about 35.0 Pa-s, or about 40.0 Pa·s, or about 45.0 Pa·s, or about 50.0 Pa·s, or about 60.0 Pa·s, or about 70.0 Pa·s, or about 80.0 Pa·s, or about 90.0 Pa·s, or about 100.0 Pa-s, or about 200.0 Pa-s, or about 300.0 Pa-s, or about 400.0 Pa-s, or about 500.0 Pa-s, or about 600.0 Pa-s, or about 700.0 Pa-s, or about 800.0 Pa·s, or about 900.0 Pa·s, or about 1,000 Pa·s, or about 2,000 Pa·s, or about 3,000 Pa·s. As described above, it can be understood that the viscosity of the hydrogel according to the present invention discloses a numerical range having any two values described above as an upper limit and a lower limit, respectively, and/or a numerical value between any two numerical values. there is.

Hereinafter, specific manufacturing examples of the present invention will be described.

An aqueous polysaccharide solution was prepared according to the formulation shown in Table 7 below and irradiated with an electron beam. As the solvent of the polysaccharide aqueous solution, only sterile water was used, and no organic solvent was added. For the electron beam, a DC-type electron beam accelerator was used.

polysaccharide Molecular Weight
(kDa) aqueous solution concentration
(w/v%) electron beam dose
(kGy) time
(s) temperature
(℃) Preparation Example 1 hyaluronic acid 5.0 0.1 5.0 150~ 39 Preparation 2 hyaluronic acid 5.0 1.0 5.0 150~ 39 Preparation 3 hyaluronic acid 5.0 5.0 5.0 150~ 39 Preparation 4 hyaluronic acid 5.0 15.0 5.0 150~ 39 Preparation 5 hyaluronic acid 5.0 20.0 5.0 150~ 39 Preparation 6 hyaluronic acid 5.0 0.1 15.0 150~ 40 Preparation 7 hyaluronic acid 5.0 1.0 15.0 150~ 40 Preparation 8 hyaluronic acid 5.0 5.0 15.0 150~ 40 Preparation 9 hyaluronic acid 5.0 15.0 15.0 150~ 40 Preparation 10 hyaluronic acid 5.0 20.0 15.0 150~ 40 Preparation 11 hyaluronic acid 5.0 0.1 30.0 150~ 45 Preparation 12 hyaluronic acid 5.0 1.0 30.0 150~ 45 Preparation 13 hyaluronic acid 5.0 5.0 30.0 150~ 45 Preparation 14 hyaluronic acid 5.0 15.0 30.0 150~ 45 Preparation 15 hyaluronic acid 5.0 20.0 30.0 150~ 45 Preparation 16 hyaluronic acid 5.0 0.1 100.0 210~ 48 Preparation 17 hyaluronic acid 5.0 1.0 100.0 210~ 48 Preparation 18 hyaluronic acid 5.0 5.0 100.0 210~ 48 Preparation 19 hyaluronic acid 5.0 15.0 100.0 210~ 48 Preparation 20 hyaluronic acid 5.0 20.0 100.0 210~ 48 Preparation 21 hyaluronic acid 5.0 0.1 150.0 300~ 48 Preparation 22 hyaluronic acid 5.0 1.0 150.0 300~ 48 Preparation 23 hyaluronic acid 5.0 8.0 150.0 300~ 48 Preparation 24 hyaluronic acid 5.0 15.0 150.0 300~ 48 Preparation 25 hyaluronic acid 5.0 20.0 150.0 300~ 48

In addition, according to the formulation shown in Table 8 below, an aqueous polysaccharide solution was prepared and irradiated with an electron beam. As the solvent of the polysaccharide aqueous solution, only sterile water was used, and no organic solvent was added. For the electron beam, a DC-type electron beam accelerator was used.

polysaccharide Molecular Weight
(kDa) aqueous solution concentration
(w/v%) electron beam dose
(kGy) time
(s) temperature
(℃) Preparation 26 Dextran 7.0 0.1 5.0 90~ 38 Preparation 27 Dextran 7.0 2.0 5.0 90~ 38 Preparation 28 Dextran 7.0 15.0 5.0 90~ 38 Preparation 29 Dextran 7.0 20.0 5.0 150~ 38 Preparation 30 Dextran 7.0 25.0 5.0 300~ 38 Preparation 31 Dextran 7.0 0.1 10.0 90~ 38 Preparation 32 Dextran 7.0 2.0 10.0 90~ 38 Preparation 33 Dextran 7.0 15.0 10.0 90~ 38 Preparation 34 Dextran 7.0 20.0 10.0 150~ 38 Preparation 35 Dextran 7.0 25.0 10.0 300~ 38 Preparation 36 Dextran 11.0 0.1 15.0 90~ 38 Preparation 37 Dextran 11.0 2.0 15.0 90~ 38 Preparation 38 Dextran 11.0 15.0 15.0 90~ 38 Preparation 39 Dextran 11.0 20.0 15.0 150~ 38 Preparation 40 Dextran 11.0 25.0 15.0 300~ 38 Preparation 41 Dextran 10.0 0.1 25.0 90~ 38 Preparation 42 Dextran 10.0 2.0 25.0 90~ 38 Preparation 43 Dextran 10.0 15.0 25.0 90~ 38 Preparation 44 Dextran 10.0 20.0 25.0 150~ 38 Preparation 45 Dextran 10.0 25.0 25.0 300~ 38

In addition, according to the formulation shown in Table 9 below, an aqueous polysaccharide solution was prepared and irradiated with an electron beam. As the solvent of the polysaccharide aqueous solution, only sterile water was used, and no organic solvent was added. For the electron beam, a DC-type electron beam accelerator was used.

polysaccharide Molecular Weight
(kDa) aqueous solution concentration
(w/v%) electron beam dose
(kGy) time
(s) temperature
(℃) Preparation 46 fucoidan 2.0 7.0 10.0 120~ 40 Preparation 47 fucoidan 2.0 10.0 20.0 120~ 40 Preparation 48 fucoidan 2.0 10.0 50.0 120~ 40 Preparation 49 fucoidan 2.0 15.0 50.0 120~ 40 Preparation 50 fucoidan 2.0 25.0 50.0 360~ 40

In addition, according to the formulation shown in Table 10 below, an aqueous polysaccharide solution was prepared and irradiated with an electron beam. As the solvent of the polysaccharide aqueous solution, only sterile water was used, and no organic solvent was added. For the electron beam, a DC-type electron beam accelerator was used.

polysaccharide Molecular Weight
(kDa) aqueous solution concentration
(w/v%) electron beam dose
(kGy) time
(s) temperature
(℃) Preparation 51 fructooligosaccharide 10.0 5.0 4.0 30~ 40 Preparation 52 fructooligosaccharide 10.0 10.0 7.0 60~ 40 Preparation 53 fructooligosaccharide 10.0 10.0 15.0 120~ 40 Preparation 54 fructooligosaccharide 10.0 15.0 15.0 120~ 40 Preparation 55 fructooligosaccharide 10.0 15.0 21.5 150~ 40 Preparation 56 fructooligosaccharide 10.0 20.0 21.5 150~ 40 Preparation 57 fructooligosaccharide 10.0 20.0 25.0 180~ 40 Preparation 58 fructooligosaccharide 10.0 20.0 30.5 180~ 40 Preparation 59 fructooligosaccharide 10.0 22.0 30.5 180~ 40 Preparation 60 fructooligosaccharide 10.0 22.0 50.0 360~ 40

In addition, according to the formulation shown in Table 11 below, an aqueous polysaccharide solution was prepared and irradiated with an electron beam. As the solvent of the polysaccharide aqueous solution, only sterile water was used, and no organic solvent was added. For the electron beam, a DC-type electron beam accelerator was used.

polysaccharide Molecular Weight
(kDa) aqueous solution concentration
(w/v%) electron beam dose
(kGy) time
(s) temperature
(℃) Preparation 61 isomaltooligosaccharide 7.0 4.0 7.0 90~ 41 Preparation 62 isomaltooligosaccharide 7.0 10.0 7.0 90~ 45 Preparation 63 isomaltooligosaccharide 7.0 11.0 7.0 90~ 45 Preparation 64 isomaltooligosaccharide 7.0 12.0 7.0 90~ 45 Preparation 65 isomaltooligosaccharide 7.0 15.0 7.0 90~ 45 Preparation 66 isomaltooligosaccharide 7.0 4.0 15.0 180~ 41 Preparation 67 isomaltooligosaccharide 7.0 10.0 15.0 180~ 45 Preparation 67 isomaltooligosaccharide 7.0 11.0 15.0 180~ 45 Preparation 69 isomaltooligosaccharide 7.0 12.0 15.0 180~ 45 Preparation 70 isomaltooligosaccharide 7.0 15.0 15.0 180~ 45

Hereinafter, the third invention will be described in detail.

[Third invention]

The method for producing a hydrogel for filler treatment according to an embodiment of the present invention may include the following steps. The following steps may be performed in alphabetical order, but the present invention is not limited thereto.

(a) adding a crosslinking agent to an aqueous solution of hyaluronic acid to prepare a hydrogel (first hydrogel) in which at least a portion of the hyaluronic acid is crosslinked

(b) sealing the hydrogel in a first pouch made of plastic material

(c) preparing a modified hydrogel (second hydrogel) by irradiating an electron-beam to the hydrogel sealed in the pouch

(d) removing the air bubbles of the hydrogel by opening the pouch and repackaging the second pouch

Unless explicitly stated otherwise, watts (W) are used as the output unit of the electron beam hereinafter, and may be expressed as energy per unit time (J/sec). Those of ordinary skill in the art to which the present invention pertains will understand by converting the unit into another unit having a dimension compatible with the unit.

As used herein, the term 'pendant' or 'pendant group' refers to an appendage. The pendant may or may not be potentially reactive. In addition, the term 'pendant crosslinked body' refers to a crosslinked body having a pendant group.

Hereinafter, each step will be described in detail.

Step (a)

The crosslinking agent is capable of crosslinking the polymer chains of hyaluronic acid through chemical bonding, and may be selected from alkyl diepoxy, diglycidyl ether, divinyl sulfone, epichlorhydrin, and the like. The crosslinking agent may be a bifunctional compound having a pair of functional groups capable of reacting with hyaluronic acid. In an exemplary embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane (1,2,7) ,8-Diepoxyoctane), 1,5-hexadiene diepoxide (1,5-Hexadiene diepoxide) and bisphenol A diglycidyl ether (Bisphenol A diglycidyl ether) may include at least one selected from the group consisting of. Preferably, the crosslinking agent may include 1,4-butanediol diglycidyl ether. More preferably, the crosslinking agent may consist only of 1,4-butanediol diglycidyl ether.

In some embodiments, the crosslinking agent is 1,4-bis(2,3-epoxypropoxy)butane (1,4-Bis(2,3-epoxypropoxy)butane), 1,4-bisglycidyloxybutane ( 1,4-Bisglycidyloxybutane), 1,2-bis(2,3-epoxypropoxy)ethylene (1,2-Bis(2,3-epoxypropoxy)ethylene), 1-(2,3-epoxypropyl)-2 ,3-epoxycyclohexane (1-(2,3-Epoxypropyl)-2,3-epoxycyclohexane) and 1,3-butadiene diepoxide may not be included. The above-mentioned crosslinking agents may not be easy to remove the pendant group through electron beam irradiation. This will be described later in detail.

When such a crosslinking agent is added and reacted with hyaluronic acid, a crosslinked product is formed and at least a portion of the hyaluronic acid can be crosslinked. The crosslinking method may be carried out according to the first and/or second invention described above, and a known method that is not limited thereto may be used, and is not particularly limited. In this case, some crosslinking agents may be at least partially unreacted.

For example, when BDDE is used as a crosslinking agent as shown in FIG. 1 , in BDDE, both epoxide groups at both ends react with hyaluronic acid to form a crosslinked product connecting both hyaluronic acid chains (see FIG. 1 ). A), some BDDEs may remain as a completely unreacted crosslinking agent because both ends of the epoxide groups do not react (B of FIG. 1). In particular, in some BDDEs, only the epoxide at one end reacts with hyaluronic acid and the epoxide group at the other end does not react, and may form an incomplete crosslinked product having a shape like a pendant group (Fig. 1). c). On the other hand, the chemical structure and the bonding structure of the hyaluronic acid main chain and the crosslinking agent in FIG. 1 are merely exemplary and the present invention is not limited thereto. Also, in the present specification, an incompletely crosslinked product, that is, a crosslinked product having a pendant group, is referred to as a 'pendant crosslinked product'.

Pendant crosslinked products can be reduced by controlling the amount or reaction time of the crosslinking agent, but inevitably occurs during crosslinking of hyaluronic acid according to the prior art. In particular, when the hydrogel is used as a filler for injection into the human body, the required physical properties are very limited, but when the reaction conditions are controlled to reduce the pendant crosslinked product, there is a problem that it cannot be applied as a hydrogel for filler treatment. As a non-limiting example, if the amount or reaction time of the crosslinking agent is increased, the formation rate of the pendant crosslinked body may be reduced, but the viscosity and elasticity are too high, so it cannot be applied as a hydrogel for filler treatment. For this reason, the pendant crosslinked body formation ratio of Juvidon, the world's No. 1 company in the filler hydrogel market, is about 10%.

On the other hand, if the formation ratio of the pendant cross-linked body is high, the hydrogel of hyaluronic acid may be softened to decrease physical properties and stability such as viscosity and elasticity, and may cause cytotoxicity or inflammation like unreacted residual cross-linking agent (Kablik J , Monheit GD, Yu LP, Chang G and Gershkovich J. Dermatol Surg 2009, 35(S1): 302-312; Sungchul C, et al. Journal of Biomedical Materials Research Part A, 2014-11-16).

Therefore, as described below, controlling the pendant cross-linked product by inducing an additional reaction of the pendant cross-linked product according to the embodiments of the present invention is a very important problem for improving the physical properties of the hydrogel and reducing toxicity, the inventors of the present invention has completed the present invention by focusing on a manufacturing method that can significantly reduce the formation ratio of a pendant crosslinked body while maintaining substantially the same level of physical properties as a conventional hydrogel for filler treatment.

In an exemplary embodiment, the hyaluronic acid may be 100 kDa or more, and the concentration of the hyaluronic acid aqueous solution may be 5 to 30 mg/mL, but the present invention is not limited thereto.

In addition, the amount of the crosslinking agent added may be 0.01 to 10 wt% compared to the aqueous solution of hyaluronic acid, and the reaction time after the addition of the crosslinking agent may be 0.5 to 8 hours, but the present invention is not limited thereto, and the molecular weight of hyaluronic acid and the amount of the crosslinking agent added , it will be clearly understood by those skilled in the art that known reaction conditions may be used.

step (b)

The hydrogel of hyaluronic acid obtained by reacting with the crosslinking agent in step (a) is placed in a first pouch made of a plastic material such as a medical pouch and sealed. In the present specification, 'sealed' means that the pouch is completely sealed so that the hydrogel is completely blocked from the outside, and also includes a state in which the hydrogel is accommodated in the partially opened pouch so that it does not flow to the outside. As a non-limiting example, the volume occupied by the hydrogel sealed in the pouch may be at least about 97%, or at least about 98%, or at least about 99% of the internal volume provided by the pouch.

The internal volume provided by the first pouch may be about 100 ml to 200 ml, or about 150 ml. The internal volume of the first pouch may be related to the energy of the electron beam in step (c), which will be described later, but the present invention is not limited thereto.

The color of the first pouch is not limited as long as it is capable of inducing additional cross-linking of the hydrogel inside by passing electron beams as described below, but preferably, a polyvinyl chloride (PVC) film having high transparency may be used.

In an exemplary embodiment, it may be preferable that the first pouch does not use a polyethylene (PE) film, a polyethylene terephthalate (PET) film, or a polypropylene (PP) film. Polyethylene, polyethylene terephthalate and polypropylene films may exhibit excellent transparency like polyvinyl chloride, but may not have sufficient durability to allow electron beams to continuously and repeatedly penetrate. That is, when a polyvinyl chloride film is used as the material of the first pouch, the pendant group or the pendant crosslinked product may be removed by irradiating an electron beam with sufficient energy in step (c) to be described later. On the other hand, when polyethylene, polyethylene terephthalate, and polypropylene film are used as the material of the first pouch, a problem such as damage to the first pouch may occur in step (c), which will be described later.

The above-described polyethylene, polyethylene terephthalate and polypropylene films may also have a sufficient thickness, but the durability problem may be somewhat solved, but as the film thickness of the first pouch increases, the transmittance of the electron beam is rapidly reduced, so that the hydrogel is partially It may be carbonized, or the process and time performed in step (c) may be complicated.

In an exemplary embodiment, the thickness of the first pouch made of polyvinyl chloride material according to the present invention may be about 0.1 mm to 0.5 mm. As described above, the polyvinyl chloride pouch has the advantage of being able to use a relatively thin pouch because it is easy for electron beams to pass therethrough.

In some embodiments, after sealing the first hydrogel in the first pouch in step (b), the method may further include aging the sealed first hydrogel. The first hydrogel prepared in step (a) maintains a relatively high temperature state during the reaction process and energy may remain. If the electron beam is irradiated through step (c) to be described later without sufficiently aging the first hydrogel, carbonization or curing of the hydrogel proceeds rather than removal of the pendant group, and the transparency of the hydrogel may be reduced.

Aging may be performed for about 10 minutes to 1 hour. If the aging time is less than 10 minutes, the effect of aging is insignificant and the above-described problem may occur. In addition, the aging may be performed in a temperature range of 25 ℃ to 30 ℃. If the aging temperature is less than 25 ℃, the viscosity and elasticity of the first hydrogel is rapidly reduced, and the effect of removing the pendant during electron beam irradiation may be insignificant. On the other hand, if the aging temperature is about 30° C. or more, or about 35° C. or more, the purpose of aging may not be achieved.

step (c)

When an electron beam is irradiated to the pouch in which the hydrogel is sealed, the electron beam can pass through the pouch to induce a reaction of the unreacted crosslinking agent in the hydrogel, and in particular, by inducing the reaction of the unreacted functional group of the pendant crosslinked product, the pendant group is at least can be partially removed. For example, when BDDE is used as a crosslinking agent as shown in FIG. 2, the unreacted epoxide group at one end of the pendant crosslinked product (A in FIG. 1) reacts with the other hyaluronic acid chain by electron beam irradiation to form both hyaluronic acid chains. It can be converted into a linking crosslinked product (B of FIG. 2). That is, by controlling the pendant cross-linked product through electron beam irradiation, the physical properties of the hydrogel can be improved and toxicity can be reduced. .

[Equation 1]

Pendant crosslinked product formation ratio = (pendant crosslinked product amount) / (crosslinked product amount)

In Formula 1, the cross-linked product means a complete cross-linked product connected to hyaluronic acid at both ends, and the amounts of the pendant cross-linked product and the cross-linked product can be expressed by the number or mass of each structure.

In the hydrogel irradiated with electron beam, as the unreacted end of the pendant crosslinked product reacted, the amount of the pendant crosslinked product decreased and the amount of the crosslinked product increased, so the pendant crosslinked product formation ratio decreased. That is, the hydrogel (second hydrogel) prepared in step (c) has a lower pendant crosslinker formation ratio than the hydrogel (first hydrogel) prepared in step (a). Preferably, the pendant cross-linkage formation ratio of the second hydrogel may be about 60% or less, or about 50% or less, or about 40% or less of the pendant cross-linkage formation ratio of the first hydrogel, but is not limited thereto, Those of ordinary skill in the art to which the present invention pertains will clearly understand that the preferred range of the formation ratio of the pendant crosslinked body may vary depending on specific manufacturing conditions. However, the present invention is intended to highlight the effect of significantly lowering the pendant formation ratio while having physical properties suitable for filler treatment on the human body.

In an exemplary embodiment, the step of irradiating the electron beam (step (c)) may include the following detailed steps.

a) Placing the first hydrogel sealed in the first pouch on a moving table

b) fixing the position of the electron beam irradiator

c) exposing the first hydrogel to an electron beam to have a predetermined period and rest period by moving the moving table

The moving table may include a turn table or a conveyor table. However, the present invention is not limited thereto, and it is not particularly limited as long as the position of the placed first hydrogel can be repeatedly, periodically, or intentionally changed. In addition, the electron beam irradiator is fixed at a specific position, and the electron beam can be irradiated only to the corresponding point. That is, the electron beam irradiator can be fixedly arranged to be irradiated to the same point regardless of the change in the position of the first hydrogel placed on the moving table.

The electron beam may be irradiated with an intensity and output of 0.1 kW to 1.0 kW. While the electron beam is irradiated to a certain point with the above intensity, the first hydrogel may be placed on a moving table and repeat passing the point where the electron beam is irradiated. In the present specification, a section in which the first hydrogel is irradiated to an electron beam is expressed as a 'period', and a section in which the first hydrogel is not exposed to an electron beam is expressed as a 'resting period'.

In an exemplary embodiment, when using an electron beam having an energy in the above-described range, the period in which the first hydrogel is irradiated (or exposed) to the electron beam may be about 0.5 seconds to 2 seconds. If the period is less than 0.5 seconds, the electron beam does not exceed the threshold energy required to contribute to the removal of the pendant crosslinked product, and thus additional crosslinking or removal of the pendant group may not be substantially performed. On the other hand, if the cycle exceeds 2 seconds, carbonization of the hydrogel may proceed and the color of the hydrogel may become opaque, rather than additional crosslinking or removal of the pendant group.

Also, in an exemplary embodiment, when using an electron beam having an energy in the above-described range, the resting period in which the electron beam is not irradiated (or not exposed) to the first hydrogel may be about 50 seconds or more. For example, the rest period may be about 50 seconds or more and 120 seconds or less, or about 50 seconds or more and 90 seconds or less, or about 50 seconds or more and 60 seconds or less. If the resting period is less than 50 seconds, carbonization of the hydrogel may proceed rather than additional crosslinking or removal of pendant groups. The upper limit of the rest period is not particularly limited, but may be about 120 seconds or less, or about 90 seconds or less, or about 60 seconds or less in terms of speed of the process.

The period and the time of the rest period may be controlled through the moving speed or rotation speed of the moving table. In addition, a plurality of sealed first hydrogels may be placed on the moving table.

That is, the detailed step c) described above may be performed by alternately repeating the cycle and the rest period. In this case, the total number of cycles may be 30 or more and 40 or less. In this case, the rest period may be a total of 29 to 39 times, but of course, the present invention is not limited thereto. If detailed step c) is performed less than 30 times, sufficient removal of the pendant crosslinked product may not be achieved, and the effect of improving physical properties and lowering toxicity may be insufficient. On the other hand, when detailed step c) is performed more than 40 times, it may be undesirable in terms of speed or easiness of the process.

As described above, by precisely controlling the irradiation time and output of the electron beam to selectively induce only the reaction of the pendant crosslinked product, the physical properties of the hydrogel can be effectively improved. For example, through this step, the second hydrogel may have increased viscosity and elasticity compared to the first hydrogel. As a specific example, the viscosity of the second hydrogel may be in the range of 400 Pa·s to 1600 Pa·s. In addition, the elasticity of the second hydrogel may be in the range of 100 Pa to 300 Pa.

Although the electron beam irradiation according to embodiments of the present invention mainly induces the reaction of the pendant crosslinker, it will be apparent that it can also induce the reaction of the completely unreacted crosslinker.

step (d)

The first pouch in which the second hydrogel irradiated with electron beams in step (c) is sealed is opened and air bubbles in the hydrogel are removed. The defoaming process may be performed through a known method. And the defoamed second hydrogel may be transferred to a second pouch and repackaged to be treated as a final product, or may be additionally subjected to a known addition process or sterilization process to be used for a filler procedure according to some embodiments.

In an exemplary embodiment, the second pouch may be formed of one selected from the group consisting of a polyethylene (PE) film, a polyethylene terephthalate (PET) film, and a polypropylene (PP) film. The second hydrogel sealed in the second pouch can be stored for a relatively long time. Therefore, it may be preferable to use a pouch that does not contain chlorine (Cl) in terms of harmlessness to the human body. In addition, the thickness of the second pouch may be thicker than the thickness of the first pouch. As described above, the first pouch is subject to electron beam irradiation, and the thickness should be selected in consideration of the electron beam irradiation intensity, etc., but the second pouch may use a pouch having a conventional thickness suitable for use in packaging. .

In some embodiments, the hydrogel for filler procedures of the present invention may comprise a generally physiologically acceptable carrier fluid, such as an isotonic buffer, particularly preferably a buffered physiological saline solution.

As a non-limiting example, the hydrogel for filler treatment of the present invention may contain 1 to 20 parts by weight of the composition for improving the persistence of hyaluronic acid in the body compared to 100 parts by weight of the composition.

The hydrogel for filler treatment of the present invention is preferably used to treat cosmetic diseases, such as wrinkles or wrinkles of the skin (eg, facial wrinkles and facial wrinkles), glabellar lines, nasolabial folds, chin wrinkles, marionette wrinkles , oral cavity, periorbital folds, fine lines around the eyes, skin depressions, scars, temples, subdermal supports of eyebrows, cheek and cheek fat pads, lacrimal gullies, nose, lips, cheeks, periorbital area, suborbital area, facial asymmetry , for the treatment of the lower jaw line and the jaw. Alternatively, it may be administered to treat therapeutic indications, such as stress incontinence, bladder-ureteral reflux, vocal fold insufficiency, vocal fold mediation.

As described above, the method for producing a hydrogel for filler treatment according to embodiments of the present invention converts an incompletely crosslinked body such as a pendant crosslinked body into a fully crosslinked body through electron beam irradiation. Even if it is used or the reaction time with the crosslinking agent is shortened, the physical properties of the hydrogel can be improved to the same or higher level.

In addition, it is possible to reduce the amount of the cross-linking agent used to obtain the desired physical properties, as well as to react and remove the non-reacted cross-linking agent that causes toxicity, such as a pendant cross-linked product, thereby remarkably reducing the toxicity of the hydrogel.

Although the physical properties of the second hydrogel prepared according to the embodiments of the present invention are not particularly limited, the amount of the crosslinking agent used in step (a) is 0.1 wt% or less compared to the aqueous hyaluronic acid solution, or the reaction between the aqueous solution of hyaluronic acid and the crosslinking agent Based on the time period of 1 hour or less, the second hydrogel has a viscosity of 400 Pa·s or more, or about 600 Pa·s or more, or about 700 Pa·s or more, or about 900 Pa·s or more, or about 1,000 It may be more than Pa·s. In addition, the second hydrogel may have an elasticity of 100 Pa or more, or about 300 Pa or more, or about 400 Pa or more, or about 500 Pa or more, or about 600 Pa or more. That is, it is suitable to be used as a hydrogel for filler treatment by exhibiting the viscosity and elasticity of the conventionally known product level, and at the same time may contain a significantly lower level of pendant crosslinked product.

Hereinafter, embodiments of the present invention will be described in more detail through specific preparation examples and experimental examples. However, the following preparation examples and experimental examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1: Preparation of the first hydrogel of cross-linked hyaluronic acid (Examples 1-1 to 1-4)

After dissolving 15 mg/mL of hyaluronic acid of about 400 kDa in 1 L of distilled water, 0.1 wt% of BDDE (1,4-butanediol diglycidyl ether) as a crosslinking agent was added at 40°C at 500 rpm. By reacting for 4 hours with stirring, a hydrogel of cross-linked hyaluronic acid was prepared (Example 1-1).

In addition, the hydrogels of Examples 1-2 to 1-4 were also prepared by varying the addition amount or reaction time of BDDE as shown in Table 12 below.

Example hyaluronic acid BDDE (wt%) Reaction time (h) Example 1-1 about 400 kDa
15 mg/mL 0.1 One Example 1-2 0.1 4 Examples 1-3 2.0 One Examples 1-4 2.0 4

Preparation Example 2: Preparation of the second hydrogel (Examples 2-1 to 2-4)

The hydrogels of Examples 1-1 to 1-4 prepared by crosslinking with the BDDE were transferred to and sealed in a medical pouch made of a transparent PVC film with a thickness of 0.3 mm, respectively. The capacity of the pouch was 150 ml. And it was stored for 15 minutes in a dark room maintained at 30 ℃.

After fixing the pouch containing the first hydrogel on the turning table, the turning table was rotated. The rotation speed was set to 1 rpm. When the rotational speed of the turning table became constant, a 0.1 kW electron beam was irradiated to a part of the turning table with a DC electron-beam accelerator. The pouch containing the first hydrogel was intermittently exposed once per minute while rotating with the turning table. And after a total of 30 rotations, the electron beam irradiation was stopped.

Then, the pouch was opened and the bubbles of the hydrogel were removed to prepare a second hydrogel of hyaluronic acid of Examples 2-1 to 2-4 as shown in Table 13 below.

Example Additional bridging target electron beam irradiation Example 2-1 Example 1-1 Rotational speed: 1rpm
Power (kW): 0.1
Using PVC film (0.3 mm) Example 2-2 Example 1-2 Example 2-3 Examples 1-3 Example 2-4 Examples 1-4

Experimental Example 1: Measurement of physical properties of Examples 1-1 to 2-4

The viscosity (shear viscosity), elasticity (elasticity) and the formation ratio of the pendant crosslinked body of the prepared Examples 1-1 to 2-4 were measured.

Viscosity was measured under shear rate 0.05 s ^-1 , 35 ℃ conditions, and elasticity was measured under frequency 1 Hz and 35 ℃ conditions. The pendant crosslinker formation ratio was measured as follows (Kablik J, Monheit GD, Yu LP, Chang G and Gershkovich J. Dermatol Surg 2009; 35(S1): 302-312).

Each sample of Examples 1-1 to 2-4 was digested with Streptomyces-derived hyaluronidase (VWR Scientific, Bridgeport, NJ) in acetate buffer at 37° C. and pH 5.0 for 72 hours to digest oligosaccharides ( oligosaccharides) were obtained. Oligosaccharides were analyzed using high-performance liquid chromatography (HPLC). For HPLC, an anionic exchange column (4x250 mm, CarboPac PA 100, Dionex Corporation, Sunnyvale, CA) was used. HPLC separates each oligosaccharide fragment according to its negative charge, and incomplete degradation of hyaluronidase causes an elution pattern according to the size of the oligosaccharide. Since hyaluronidase cannot decompose the crosslinked product, observing a peak eluting at a retention time greater than or equal to octasaccharide indicates the presence of a specific crosslinked oligosaccharide. Thus, the cross-linked ratio is determined as the sum of the delayed eluted peaks, and since the total peak area is proportional to the concentration of each fragment, the relative proportion of cross-linked or pendant cross-linked parts can be determined.

By substituting the obtained data into Equations 2 and 3 below, the pendant crosslinked body formation ratio was obtained.

[Equation 2]

Total degree of modification (%) = proportion of crosslinked product (%) + proportion of pendant crosslinked product (%)

[Equation 3]

Pendant crosslinked body formation ratio = pendant crosslinked body ratio (%) / crosslinked body ratio (%)

The measured viscosity, elasticity, and pendant crosslinked product formation ratio are shown in Table 14 below.

Example Viscosity (Pa s) Elasticity (Pa) Pendant crosslink formation ratio Example 1-1 409.1 103.5 1.9 Example 1-2 498.3 154.2 1.6 Examples 1-3 741.0 210.3 2.1 Examples 1-4 888.9 339.8 1.7 Example 2-1 701.2 306.3 0.9 Example 2-2 930.2 439.1 0.5 Example 2-3 1243.7 590.7 0.8 Example 2-4 1397.6 686.0 0.6

Referring to Table 14, it can be seen that the greater the amount of BDDE added and the longer the reaction time, the higher the viscosity and elasticity, and the lower the pendant cross-linked product formation ratio. Examples 1-1 to 1-4 It can be seen that Examples 2-1 to 2-4 obtained by further crosslinking by electron beam irradiation have significantly improved viscosity and elasticity compared to those obtained before additional crosslinking. Considering that the formation ratio of the pendant cross-linked body in Examples 2-1 to 2-4 is significantly lower than before the additional cross-linking, it can be considered that such improvement in viscosity and elasticity is due to the reaction of the pendant cross-linked body by electron beam irradiation. there is.

Through this, even if a relatively small amount of a crosslinking agent such as BDDE is used when crosslinking hyaluronic acid or the reaction time with the crosslinking agent is relatively short, additional electron beam irradiation induces a reaction of the pendant crosslinker to additionally crosslink the hyaluronic acid. It can be seen that a level or excellent level of physical properties can be secured.

Preparation Example 3: Change of irradiation time and output of electron beams (Examples 3-1 to 3-12)

A hydrogel was prepared in the same manner as in Example 1-2, and transferred to a medical pouch made of a transparent PVC film with a thickness of 0.3 mm and sealed. And in the same manner as in Preparation Example 2, the pouch containing the hydrogel was intermittently exposed to the electron beam using a turning table, and the rotation speed was controlled to vary the total irradiation time and output as shown in Table 15 below. After the electron beam irradiation was finished, the pouch was opened and the bubbles of the hydrogel were removed to prepare additional cross-linked hyaluronic acid hydrogels of Examples 3-1 to 3-12 as shown in Table 15 below.

Example Irradiation time (sec) Power (kW) pouch Example 3-1 about 0.3 0.01 Using PVC film (0.3 mm) Example 3-2 about 0.3 0.1 Example 3-3 about 0.3 1.0 Example 3-4 about 1 0.01 Example 3-5 (Example 2-2) about 1 0.1 Example 3-6 about 1 1.0 Example 3-7 about 2 0.01 Example 3-8 about 2 0.1 Example 3-9 about 2 1.0 Example 3-10 about 10 0.01 Example 3-11 about 10 0.1 Example 3-12 about 10 1.0

Experimental Example 2: Measurement of physical properties of Examples 3-1 to 3-12

The viscosity, elasticity, and pendant crosslinked body formation ratio of the prepared Examples 3-1 to 3-12 were measured in the same manner as in Experimental Example 1, and the results are shown in Table 16 below.

Example Viscosity (Pa s) Elasticity (Pa) Pendant crosslink formation ratio Example 3-1 512.6 151.1 0.8 Example 3-2 527.0 154.9 0.8 Example 3-3 683.5 176.4 0.7 Example 3-4 885.0 427.2 0.4 Example 3-5 (Example 2-2) 930.2 439.1 0.5 Example 3-6 1132.3 636.0 0.2 Example 3-7 1026.7 551.8 0.4 Example 3-8 1073.0 605.1 0.4 Example 3-9 1273.0 725.1 0.2 Example 3-10 N/A (physical properties cannot be measured due to hydrogel combustion) Example 3-11 N/A (physical properties cannot be measured due to hydrogel combustion) Example 3-12 N/A (physical properties cannot be measured due to hydrogel combustion)

Referring to Table 16, the longer the electron beam irradiation time and the higher the output, the better the physical properties such as viscosity and elasticity. When the output is less than 0.1 kW, the change in the formation ratio of the pendant crosslinked body according to the increase in output is small, so the efficiency of increasing the physical properties is also not good. On the other hand, when the irradiation time was 10 seconds, there was a problem that the hydrogel was partially burned by the electron beam.

Preparation Example 4: Change of pouch material (Examples 4-1 to 4-4)

A hydrogel was prepared in the same manner as in Example 1-2, and transferred to a medical pouch composed of a transparent film with a thickness of 0.3 mm of various materials as shown in Table 17 below and sealed. And the electron beam was irradiated in the same manner as in Preparation Example 2.

Example Pouch material (0.5 mm) electron beam irradiation Example 4-1 (Example 2-2) PVC film Good Example 4-2 PE film pouch burst Example 4-3 PET film pouch burst Example 4-4 PP film pouch burst

Preparation Example 5: Change in the thickness of the pouch (Examples 5-1 to 5-4)

A hydrogel was prepared in the same manner as in Example 1-2, and transferred to a medical pouch composed of films of various materials and thicknesses as shown in Table 18 below and sealed. And the electron beam was irradiated in the same manner as in Preparation Example 2.

Example pouch material Pouch thickness (mm) electron beam irradiation Example 5-1 (Example 2-2) PVC 0.3 Rotational speed: 1rpm
Power (kW): 0.1
Using PVC film (0.3 mm) Example 5-2 PVC 0.7 Example 5-3 PE film 1.0 Example 5-4 PET film 1.0 Example 5-5 PP film 1.0

Experimental Example 4: Measurement of physical properties of Examples 5-1 to 5-5

The viscosity, elasticity, and pendant crosslinked body formation ratio of the prepared Examples 5-1 to 5-5 were measured in the same manner as in Experimental Example 1, and the results are shown in Table 19 below.

Example Viscosity (Pa s) Elasticity (Pa) Pendant crosslink formation ratio Example 5-1 (Example 2-2) 930.2 439.1 0.5 Example 5-2 895.7 490.1 0.4 Example 5-3 719.3 368.2 0.7 Example 5-4 604.6 298.1 0.5 Example 5-5 749.1 327.4 0.4

Referring to Table 19, it can be seen that the physical properties of Example 5-1 are the best. Example 5-2, which uses a PVC film in the same manner as in Example 5-1 but with an increased thickness, also increases physical properties, but it can be seen that it is significantly lower than that of Example 5-1.

In the case of Examples 5-3 to 5-5, in which the thickness of the pouch film is reinforced so that the pouch film does not burst, it can be seen that the effect of increasing the physical properties is insignificant.

Preparation Example 6: Change of type of crosslinking agent (Examples 6-1 to 6-4)

In the same manner as in Example 1-2, the hydrogel was transferred to a medical pouch made of a transparent PVC film with a thickness of 0.3 mm using various crosslinking agents and sealed. And the electron beam was irradiated in the same manner as in Preparation Example 2.

Example crosslinking agent Example 6-1 (Example 2-2) BDDE Example 6-2 1,4-Bis(2,3-epoxypropoxy)butane Example 6-3 1-(2,3-Epoxypropyl)-2,3-epoxycyclohexane Example 6-4 1,3-Butadiene diepoxide

Experimental Example 5: Measurement of physical properties of Examples 6-1 to 6-4

The viscosity and elasticity of the prepared Examples 5-1 to 5-5 were measured in the same manner as in Experimental Example 1, and the results are shown in Table 21 below.

Example Viscosity (Pa s) Elasticity (Pa) Example 6-1 (Example 2-2) 930.2 439.1 Example 6-2 610.6 307.0 Example 6-3 678.4 316.4 Example 6-4 712.5 310.5

Referring to Table 21, it can be seen that the physical properties of Example 6-1 are the best. In the case of other Examples 6-2 to 6-4, it can be seen that the synergistic effect of physical properties is insignificant. Although the present invention is not limited by any theory, it may be because at least the additional reaction of the crosslinking agent does not proceed according to the pendant removal process conditions used in the present invention.

As described above, it has been reported that a hydrogel is formed by adding a crosslinking agent such as BDDE to a biocompatible polymer such as hyaluronic acid. However, a crosslinking agent such as BDDE has a limitation in that at least a part thereof is unreacted to form a pendant crosslinked product that causes deterioration of hydrogel properties and cytotoxicity. came to the conclusion of the invention.

According to the embodiments of the present invention, since the unreacted crosslinking agent, in particular, the incomplete crosslinked product such as a pendant crosslinked product is converted into a complete crosslinked body to induce additional crosslinking of hyaluronic acid, a small amount of the crosslinking agent is used or Even if the reaction time with the crosslinking agent is shortened, the physical properties of the hydrogel can be improved to the same or higher level. In addition, it is possible to reduce the amount of cross-linking agent used to obtain desired physical properties and also to react and remove unreacted cross-linking agents that cause toxicity, such as pendant cross-linkers, so that the toxicity of the hydrogel can be significantly reduced.

In the above, the embodiment of the present invention has been mainly described, but this is only an example and does not limit the present invention. It will be appreciated that various modifications and applications not exemplified above are possible.

Therefore, it should be understood that the scope of the present invention includes changes, equivalents or substitutes of the technical ideas exemplified above. For example, each component specifically shown in the embodiment of the present invention may be implemented by modification. And differences related to such modifications and applications should be construed as being included in the scope of the present invention defined in the appended claims.

Claims (1)

As a hydrogel for filler treatment,
preparing a first hydrogel by irradiating a first electron beam to crosslink an aqueous solution of hyaluronic acid; and
A hydrogel prepared by irradiating a second electron beam to at least partially remove the pendant crosslinked product of the first hydrogel to form a second hydrogel.

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