KR20210068683A - Method of preparing biocompatibility particle polymer using electron-beam cross linking - Google Patents

Abstract

The present invention relates to a method for preparing a hydrogel by irradiating an electron beam on polysaccharides, and a hydrogel prepared therefrom, and more particularly, to a method for preparing a particle type hydrogel by inducing crosslinking without substantially adding a crosslinking agent to polysaccharides such as hyaluronic acid, etc., and to a particle type hydrogel prepared therefrom. For example, the present invention provides a method for preparing a microgel for filler treatment, which comprises a step of irradiating an electron beam at a dose of 5 to 10kGy with pulses or impulses to 18 to 25 w/v% of a first aqueous solution of hyaluronic acid to at least partially crosslink the hyaluronic acid.

Description

Manufacturing method of biocompatible particulate polymer using electron beam crosslinking {METHOD OF PREPARING BIOCOMPATIBILITY PARTICLE POLYMER USING ELECTRON-BEAM CROSS LINKING}

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.

Hyaluronic acid is one of the complex negatively charged polysaccharides, and is a high molecular compound composed of N-acetylglucosamine and glucuronic acid, and is present in connective tissue, subcutaneous tissue, nerve tissue or joint synovial fluid in vivo. Because of its excellent biocompatibility, hyaluronic acid is extracted from microbial fermentation of Streptococcus genus or chicken fodder, etc., and is used not only as a cosmetic additive but also as a joint cavity injection, anti-adhesion agent, and 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 chemically cross-link it to extend the decomposition period in the body.

In other words, in order to increase the chemical structural stability of hyaluronic acid and to slow the decomposition in the living body to maintain the physical properties for a long period, 1,4-butanediol diglycidyl ether (BDDE), etc. Techniques for stabilizing hyaluronic acid by adding a crosslinking agent have been developed, among which it is known that the maximum retention period in vivo of existing crosslinked hyaluronic acid and crosslinked hyaluronic acid derivatives is about 6 to 24 months.

However, high viscoelastic cross-linked hyaluronic acid obtained by simply increasing the amount of cross-linking agent in order to enhance the residence period of hyaluronic acid in vivo may cause an inflammatory reaction by decomposing or unreacted residual cross-linking agent components in vivo causing toxicity. There is a problem that there is

In addition, in order to remove the residual crosslinking agent, an additional removal process is required, which has a problem of causing inefficiency in the process of manufacturing the hyaluronic acid filler.

On the other hand, as described above, attempts have been made to prepare a hydrogel by crosslinking polysaccharides such as hyaluronic acid. The prepared hydrogel has the advantage of excellent biocompatibility. Hydrogels (hydrogels) have a three-dimensional network structure in which polymers are cross-linked and have swelling properties that absorb fluids from the outside. Accordingly, it has a property of loading a therapeutic agent as well as a biological fluid derived from a living tissue or the like. In addition, since it is softer than other synthetic polymers, it is widely studied in medical and pharmaceutical fields.

For example, a hydrogel having a nano size or a micro size has good flowability and can absorb or load drugs. In addition, the hydrogel can self-decompose and has the advantage of being able to diversify the formulation. Such a hydrogel may be used as a dressing, a drug delivery carrier, a lens, cartilage, an anti-adhesion agent, a medical/non-medical filler, and the like. In addition, the loading body loaded on the hydrogel is not particularly limited, but may be a fluorescent material, a radioactive isotope, a contrast agent for magnetic resonance imaging or computed tomography, an ultrasound contrast agent, an anticancer agent, an anti-inflammatory agent, and the like.

Korean Patent Publication No. 10-2019-0067653 (Patent Document 2) Korean Patent Publication No. 10-2018-0096562

An object of the present invention to solve such a problem is to irradiate hyaluronic acid with an electron-beam to cause a cross-linking reaction without a separate cross-linking agent, thereby preventing problems caused by the addition of a toxic cross-linking agent.

In addition, it is to provide a hydrogel having a level of viscosity and in vivo stability that can be used as an injectable filler composition by preparing a micro-sized hydrogel having high viscosity and large particles by crosslinking hyaluronic acid under specific conditions.

In addition, it is an object to provide a method for producing a microgel in which an additional process for removing the unreacted residual cross-linking agent can be omitted by using a cross-linking agent with irradiation of electron beams but using a biocompatible cross-linking agent.

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 a method for producing a microgel for filler treatment according to an embodiment of the present invention for solving the above problems, an electron beam having a dose of 5 kGy to 10 kGy is pulsed or impulsed in a first aqueous hyaluronic acid solution of 18 w/v% to 25 w/v%. and irradiating to at least partially crosslink the hyaluronic acid.

In addition, when the electron beam is irradiated with pulses or impulses, the irradiation period may be 5 seconds to 10 seconds.

When the electron beam is irradiated with a pulse, the irradiation time may be 3 seconds or less.

In addition, the total irradiation time of the electron beam may be 10 minutes to 60 minutes.

Here, the step of crosslinking the hyaluronic acid may be performed at a temperature of 50 °C to 60 °C.

In addition, the hyaluronic acid may have a molecular weight of 100 kDa or more and 500 kDa or less.

In some embodiments, after starting the irradiation of the electron beam, the method may further include mixing a second aqueous hyaluronic acid solution having a higher concentration than the first aqueous hyaluronic acid solution.

Here, the upper limit of the concentration of the second aqueous hyaluronic acid solution may be 30w/v%.

The stirring speed of the first aqueous solution of hyaluronic acid and the second aqueous solution of hyaluronic acid may be 500 rpm to 1,000 rpm.

In addition, the irradiation of the electron beam may be continuously performed simultaneously with the mixing of the second aqueous hyaluronic acid solution.

The first aqueous solution of hyaluronic acid is 0.1wt% to 0.5wt% of a genipine derivative based on the weight of hyaluronic acid, and may further include a genipine derivative represented by the following Chemical Formula 1.

[Formula 1]

On the other hand, the first aqueous solution of hyaluronic acid may contain substantially no genipin.

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

According to embodiments of the present invention, by irradiating hyaluronic acid with an electron beam to cause a crosslinking reaction without a separate crosslinking agent, a problem caused by the addition of a toxic crosslinking agent can be prevented in advance.

In addition, it is possible to provide a hydrogel having a level of viscosity that can be used as an injectable filler composition by directly preparing a micro-sized granular hydrogel by crosslinking hyaluronic acid under certain conditions.

In addition, it is easy to control the particle size through the subsequent process, for example, it is possible to provide a hydrogel having a particle size of a micro-sized level that can be used for a filler procedure.

In addition, by using the biocompatible crosslinking agent together with the irradiation of electron beams, there is an advantage that an additional process for removing the unreacted residual crosslinking agent is unnecessary.

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 result of confirming the particle size of a microgel prepared according to Example 4 of the present invention by DLS.
2 is a result of checking the particle size of microgels prepared according to Example 5-1, Example 5-2, and Example 5-6 of the present invention by DLS.
3 is a result of checking the particle size of microgels prepared according to Example 6-1, Example 6-2, and Example 6-3 of the present invention by DLS.
4 is a result of checking the particle size of microgels prepared according to Examples 7-1 and 7-2 of the present invention by DLS.

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.

In the present specification, the dose unit of the electron beam is 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.

Hereinafter, the present invention will be described in detail. The first invention and the second 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 and the second invention, it is applied only to the present invention, and technical ideas that do not conflict with each other can be commonly applied to both inventions. Those of ordinary skill in the art will be able to understand easily.

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.

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, thereby solving the problem of causing toxicity to the human body by remaining unreacted 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, furthermore, a 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 become smaller due to the decomposition of hyaluronic acid, and finally the viscosity of the gel may be lowered. On the other hand, if the dose of the electron beam is less than 5 kGy, aggregation or sticking between particles, and further, the formation of bulk gel may occur, which is not preferable.

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 the form of pulses or impulses, 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 was dissolved in water to prepare 5, 15 and 30 w/v% aqueous solutions, and electron beams were 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 the dose of the electron beam 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. 1, and in particular, the concentration of 22w/v% is the most 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. Also, 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 by 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 As in Example 7-2, it can be seen that if the stirring speed is too high (2000 rpm) when mixing, the particles are pulverized, which is not preferable.

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 an aqueous solution of hyaluronic acid, wherein the aqueous solution of hyaluronic acid is a biocompatible crosslinking agent, Genipin (Sigma-Aldrich), and is represented by the following Chemical 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 induce 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 present specification also discloses numerical ranges having any two numerical values as an upper limit and a lower limit, 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. have.

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. have.

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. have.

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, the irradiation of the electron beam may be started 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 the electron beam for crosslinking of the polysaccharide, the dose rate of the electron beam 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, maintained for a predetermined time, and then 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 1800 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, antiallergic 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. have.

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

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)

A method for producing a microgel for filler treatment, comprising the step of at least partially crosslinking hyaluronic acid by irradiating an electron beam with a dose of 5 kGy to 10 kGy to the first aqueous hyaluronic acid solution.

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