KR20210068682A - Method of preparing bulk polymer using hyaluronic acid solution and cross linked polymer prepared therefrom - 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 bulk type hydrogel by inducing crosslinking without substantially adding a crosslinking agent to polysaccharides such as hyaluronic acid, etc., and to a bulk type hydrogel prepared therefrom. For example, the present invention provides a method for preparing muk-gel for filler treatment, which comprises the steps of: preparing a bulk hydrogel by irradiating an electron beam to an aqueous solution of 10-20 w/v% hyaluronic acid at a dose of 0.5-5 kGy for 30 seconds to 5 minutes to crosslink the hyaluronic acid; and removing air bubbles inside the bulk hydrogel.

Description

Method for producing a bulk polymer using an aqueous hyaluronic acid solution and a crosslinked polymer prepared therefrom {METHOD OF PREPARING BULK POLYMER USING HYALURONIC ACID SOLUTION AND CROSS LINKED POLYMER PREPARED THEREFROM}

According to one aspect of the present invention, the present invention relates to a method for producing a Merck gel by crosslinking hyaluronic acid, specifically, a Merck gel 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 Merck gel 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 bulk hydrogel by inducing crosslinking without substantially adding a crosslinking agent to polysaccharides such as hyaluronic acid, and to a bulk hydrogel 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, by crosslinking hyaluronic acid under specific conditions to produce a bulk hydrogel having high viscosity and large particles, a hydrogel having a level of viscosity and in vivo stability that can be used as an injectable filler composition is provided. .

In addition, it is to provide a method for manufacturing Merck Gel that uses a crosslinking agent together with electron beam irradiation, but an additional process for removing the unreacted residual crosslinking agent can be omitted by using a biocompatible crosslinking 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 manufacturing a Merck gel for filler treatment according to an embodiment of the present invention for solving the above problems, an electron-beam is applied to 10-20 w/v% hyaluronic acid aqueous solution for 30 seconds to 5 seconds. preparing a bulk hydrogel by crosslinking hyaluronic acid by irradiating it with a dose of 0.5 to 5 kGy per minute; and removing air bubbles inside the bulk hydrogel.

The irradiation of the electron beam is a first irradiation step in which the dose rate is gradually increased for a first time, followed by the first irradiation step, a second irradiation in which the dose rate is gradually decreased for a second time longer than the first time and a third irradiation step in which the dose rate is gradually increased and irradiated for a third time shorter than the second time following the second irradiation step.

Alternatively, the irradiation of the electron beam is a first irradiation step in which the dose rate is gradually decreased for a first time, followed by the first irradiation step, and the dose rate is gradually increased for a second time shorter than the first time. The second irradiation step, and following the second irradiation step, may include a third irradiation step in which the dose rate is gradually decreased and irradiated for a third time longer than the second time.

In some embodiments, between the step of preparing the bulk hydrogel and the step of removing the air bubbles, after adding 1 to 20 wt% of 1 to 20 wt% of a low molecular weight hyaluronic acid aqueous solution to the bulk hydrogel compared to the hydrogel The method may further include preparing an additional cross-linked bulk hydrogel by irradiating an electron beam with a dose of 1 to 5 kGy for 3 seconds to 20 seconds.

In the step of preparing the bulk hydrogel, an electron beam is applied to an aqueous solution containing 10-20 w/v% hyaluronic acid and 0.1 to 1 wt% of an additional material compared to hyaluronic acid in a dose of 0.5-5 kGy for 30 seconds to 5 minutes. After irradiation, maintaining a temperature of 30° C. to 42° C. and stirring for 8 hours to 16 hours may be a step of crosslinking hyaluronic acid to prepare a bulk hydrogel.

In addition, the additional material may include genipin and/or a derivative of genipin represented by Formula 1 below.

[Formula 1]

The manufacturing method of the hydrogel for filler treatment according to an embodiment of the present invention for solving the other problem includes the steps of preparing the Merck gel prepared through the above-described method, and the bulk hydrogel at 1,000 rpm to 5,000 rpm It may include the step of preparing a hydrogel having micro-sized particles by grinding at a speed of 1 to 5 minutes.

Here, the viscosity of the hydrogel prepared in the step of preparing the bulk hydrogel may be 250 ~ 1,000 Pa·s.

In addition, the hydrogel prepared in the step of preparing the hydrogel having the micro-sized particles may have an average diameter of 50 μm to 700 μm, and a viscosity of 15 to 100 Pa·s.

In some embodiments, in the step of preparing the hydrogel having the micro-sized particles, when the average diameter of the hydrogel particles is 50 μm or less, 50 to 1-5 w/v% of 5-30 kDa low molecular weight hyaluronic acid aqueous solution To obtain a hydrogel having nano-sized particles produced by irradiating an electron beam with a dose of ~200 kGy, add 1-50 wt% to the hydrogel having micro-sized particles, and then apply an electron beam for 30 seconds to 5 minutes 10 ~ A step of irradiating to a dose of 30 kGy, or adding 0.1 to 1 wt% of a biocompatible crosslinking agent to the hydrogel having the micro-sized particles, maintaining the temperature at 30°C to 42°C, and stirring for 1 hour to 8 hours It may include further steps.

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 crosslinking hyaluronic acid under certain conditions to prepare a bulk hydrogel having a high viscosity and then pulverizing it.

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 the hyaluronic acid hydrogel prepared according to the embodiments of the present invention by DLS.
2 is a result of confirming the particle size of the hyaluronic acid hydrogel prepared according to other embodiments 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.

As used herein, the term 'Muk-gel' refers to a state in which a network structure is formed and has viscoelasticity, but is not granulated and has the shape of a container or reactor in which the gel is placed. I mean gel. In this specification, the term 'Merck gel' may be used interchangeably with 'bulk gel'.

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 of manufacturing a Merck gel for filler treatment according to an embodiment of the present invention (ie, an embodiment of the first invention) may include the following steps. The following steps may be performed in the order of letters and numbers.

(a-1) 10-20 w/v% hyaluronic acid aqueous solution is irradiated with an electron-beam at a dose of 0.5-5 kGy for 30 seconds to 5 minutes to crosslink the hyaluronic acid to bulk (bulk) Preparing a hydrogel (hydrogel)

(b) removing air bubbles inside the bulk hydrogel

In some embodiments, the method for manufacturing a Merck gel for filler treatment according to the present invention may further include the following steps. The following steps may be performed in the order of letters and numbers together with the steps described above.

(a-2) After adding an aqueous solution of 1 to 80 kDa low molecular weight hyaluronic acid in an amount of 1 to 20 wt% compared to the hydrogel to the bulk hydrogel, an electron beam is irradiated to a dose of 1 to 5 kGy for 3 seconds to 20 seconds for additional crosslinking Preparing the bulk hydrogel

Also, in some embodiments, the method for producing a hydrogel for filler treatment according to the present invention may further include the following steps. The following steps may be performed in the order of letters and numbers together with the steps described above.

(c-1) pulverizing the bulk hydrogel at a speed of 1,000 rpm to 5,000 rpm for 1 minute to 5 minutes to prepare a hydrogel having micro-sized particles

(c-2) When the average diameter of the hydrogel particles in step (c-1) is 50 μm or less, 1-5 w/v% of 5-30 kDa low molecular weight hyaluronic acid aqueous solution is subjected to a 50-200 kGy dose of electron beam To obtain a hydrogel having nano-sized particles produced by irradiation, add 1-50 wt% to the hydrogel having micro-sized particles, and then irradiate an electron beam at a dose of 10-30 kGy for 30 seconds to 5 minutes Step, or adding 0.1 to 1 wt% of a biocompatible crosslinking agent to the hydrogel having the micro-sized particles, maintaining a temperature of 30°C to 42°C, and stirring for 1 hour to 8 hours

Steps (a-1) and (a-2)

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 Merck gel 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 Merck gel further comprises a crosslinking agent or a crosslinking aid 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 bulk hydrogel produced by crosslinking through the step (a-1) and/or the step (a-2) is a high-viscosity gel having a viscosity of 250 to 1,000 Pa s at 1 Hz when measured with a rheometer, It may have an appropriate viscosity and particle size to be pulverized in step (c-1) to prepare a hydrogel composed of uniform micro particles.

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 10 w/v%, bulking may not occur sufficiently, and if it is higher than 20 w/v%, the degree of bulk according to the increase in concentration is insignificant, which may be uneconomical. Alternatively, gelation is too advanced, so injection using an extrusion mechanism is difficult, and it cannot be used for filler treatment.

In addition, if the dose of the electron beam is out of 0.5 kGy to 5 kGy or the total irradiation time is out of 30 seconds to 5 minutes, crosslinking may not occur sufficiently or the particle size may be reduced due to the decomposition of hyaluronic acid, thereby lowering the viscosity. For example, when the dose of the electron beam exceeds 5 kGy, specifically, when the dose is about 10 kGy or more, or 15 kGy or more, or about 20 kGy or more, the decomposition of Merck Gel may occur. Furthermore, the local viscosity of the Merck gel may vary and the shape of the Merck gel may not be maintained. For another example, if the irradiation time exceeds 5 minutes, the Merck Gel may be decomposed. In addition, the local viscosity of Merck Gel may vary, and the shape of the Merck Gel may not be maintained. The dose rate (Gy/s) during the irradiation time may be constant or may vary.

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 10-20 w/v% hyaluronic acid aqueous solution may be 100 kDa or more, preferably 200 kDa or more.

In an exemplary embodiment, a change in the dose rate in the irradiation of the electron beam in step (a-1) may be accompanied.

That is, the step (a-1) is a first irradiation step in which the dose rate is gradually increased for the first time, and immediately following the first irradiation step, the dose rate is gradually decreased for a second time longer than the first time, A second irradiation step of being irradiated, and a third irradiation step of irradiating with a dose rate gradually increasing for a third time shorter than the second time immediately following the second irradiation step.

Alternatively, the step (a-1) is a first irradiation step in which the dose rate is gradually decreased for a first time and is irradiated, immediately following the first irradiation step, and the dose rate is gradually increased for a second time shorter than the first time. The second irradiation step, and immediately following the second irradiation step, the dose rate is gradually decreased for a third time longer than the second time, it may be carried out including a third irradiation step irradiated.

Here, the minimum and maximum doses of the electron beam during the entire irradiation time may be in the range of 0.5 kGy to 5 kGy.

In other words, the dose rate of the electron beam may be increased and decreased by repeating the increase and decrease once or more, preferably twice or more, more preferably three times or more, and still more preferably five times or more. In addition, the time of the section in which the dose rate of the electron beam is increased may be shorter than, or at least equal to, the time of the section in which the dose rate of the electron beam is decreased. For example, the time of the section in which the dose rate of the electron beam is increased may be about 5 seconds, and the time of the section in which the dose rate of the electron beam is decreased may be about 7 seconds, but the present invention is not limited thereto. As a non-limiting example, the difference between the total dose in the section in which the dose rate increases and the total dose in the section in which the dose rate decreases is substantially the same, or has a difference of about 20%, or about 30%, or about 40%. can It is desirable that the difference between the total dose in the section where the dose rate increases and the total dose in the section where the dose rate decreases is 50% or less. If the difference in dose is too large, the physical properties of the cross-linked hydrogel may be unstable.

Although it may be affected by the concentration of hyaluronic acid aqueous solution, etc., in some experiments, when electron beam is irradiated at a constant dose rate, Merck Gel has an approximately homogeneous viscosity and cannot maintain its shape, and the viscosity is partially or locally different. It could not be maintained in this perfect shape, and it was confirmed that it was partially flowing.

On the other hand, if the dose rate of the electron beam is repeatedly increased and decreased, this problem can be remarkably improved, and the present invention has been completed by confirming that it is suitable for use as a hydrogel for filler treatment.

On the other hand, in some embodiments, the aqueous solution of step (a-1) may be carried out to further include genipin and/or a derivative of genipin.

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, it is possible to increase the crosslinking rate of the hydrogel. The genipin derivative is represented by the following Chemical Formula 1 and may have an epoxy group.

[Formula 1]

Genipine or genipine derivatives can form intermolecular cross-links with hyaluronic acid. On the other hand, although the present invention is not limited thereto, cross-linking between genipine/genipine derivatives may not be substantially made.

The content range of genipine alone, genipine derivative alone, or a mixture of genipine and genipine derivative may be about 0.1 wt% to 1.0 wt% compared to hyaluronic acid. If the content of genipine and/or a genipine derivative exceeds the above range, the prepared Merck gel may not be transparent and opaque, and/or the viscosity may be too high, making it unsuitable for filler treatment.

In addition, in the crosslinking of hyaluronic acid, crosslinking may be performed while maintaining a temperature of about 30° C. to 42° C. and stirring for 8 hours to 16 hours after irradiation with an electron beam.

Meanwhile, in some embodiments, the step (a-2) may be performed after the step (a-1). That is, additional crosslinking can be performed by adding an additional aqueous hyaluronic acid solution to the bulk hydrogel prepared by crosslinking the aqueous hyaluronic acid solution using an electron beam.

The additional aqueous hyaluronic acid solution may include low molecular weight hyaluronic acid compared to the hyaluronic acid used in step (a-1). For example, hyaluronic acid of about 1 kDa to 80 kDa may be used.

In addition, in step (a-2), the electron beam may be irradiated with the same or greater dose as in step (a-1). For example, it may be irradiated with a dose of about 1 kGy to 5 kGy, or about 2 kGy to 5 kGy, or about 3 kGy to 5 kGy. As a non-limiting example, the dose rate of the electron beam may be irradiated to be approximately constant without changing.

On the other hand, step (a-2) may be performed for a shorter time than step (a-1). The irradiation time may be about 3 seconds to 20 seconds, but the present invention is not limited thereto, and may be appropriately selected in consideration of the physical properties of the hydrogel to be prepared.

Through step (a-2), it is possible to obtain a more highly viscous Merck gel, that is, a bulk gel with excellent elasticity. Through this, when the Merck gel is pulverized as in step (c-1), which will be described later, there is an advantage that the particle size size control is easy and the ease of operation can be improved.

The viscosity of the Merck gel prepared through the aforementioned step (a-1) and/or step (a-2) is about 15 Pa·s to 1,000 Pa·s, or about 700 Pa·s or more (based on Rheometer 1Hz). can

step (b)

The prepared Merck gel may be subjected to a defoaming process. The defoaming process can use a well-known method made with respect to a hydrogel. For example, it may be carried out by adding a defoaming agent, or through a physical/mechanical method.

Steps (c-1) and (c-2)

Step (c-1) may be performed using a homogenizer. In this case, the grinding speed may be 1,000 rpm to 5,000 rpm. If it is out of the above range, a uniform particulate hydrogel cannot be obtained. This may be because the viscosity of Merck Gel is very high, but the present invention is not limited to any theory. In addition, the grinding time may be about 1 to 5 minutes. Likewise, grinding time can affect the particle size uniformity of micro-sized particles.

As described above, when Merck gel and a micro-sized hydrogel obtained by pulverizing the same are used for filler treatment, particle size uniformity is a very important factor. If the particle size is not uniform, side effects may occur after the procedure, or the procedure may be difficult due to difficulty in controlling the viscosity.

The micro-sized particles constituting the hydrogel prepared by pulverizing the Merck gel may have an average diameter of about 50 μm to 700 μm, or about 100 μm to 600 μm, or about 150 μm to 500 μm, and the viscosity thereof is When measured with a rheometer, it can be 15 to 100 Pa·s at 1 Hz. The present invention has the advantage of being easy to apply for filler treatment by controlling the particulate hydrogel to the particle size and the viscosity range.

In some embodiments, (c-1) when the average diameter of the hydrogel particles prepared in the above step is 50㎛ or less, step (c-2) may be additionally performed.

That is, to obtain a hydrogel having nano-sized particles produced by irradiating an electron beam with a dose of 50 to 200 kGy to a separately prepared 1-5 w/v% 5-30 kDa low-molecular hyaluronic acid aqueous solution, and this (c-1 ), after adding 1 to 50 wt% to the hydrogel prepared in step, electron beams can be irradiated to a dose of 10 to 30 kGy for 30 seconds to 5 minutes.

Alternatively, 0.1 to 1 wt% of a biocompatible crosslinking agent is added to the hydrogel prepared in step (c-1), and a step of stirring while maintaining a temperature of 30° C. to 42° C. may be performed for 1 to 8 hours. The biocompatible crosslinking agent may include the aforementioned genipine and/or genipine derivative.

When the particle size of the hydrogel prepared in step (c-1) is small and does not exhibit an appropriate viscosity, and thus is inappropriate to use as a Merck gel for filler treatment, the desired product by further proceeding with the same process as in step (c-2) A hydrogel having a particle size and viscosity can be prepared. That is, the inventor of the present invention focused on the ability to manufacture a hydrogel having the most suitable viscosity and particle size for use in filler procedures through post-processing process control along with the degree of crosslinking, viscosity, elasticity, etc. of Merck gel. came to the conclusion of the invention.

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 Merckgel and/or 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.

In an exemplary embodiment, the Merck gel and/or 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 relative to 100 parts by weight of the composition.

The Merck gel and hydrogel for filler treatment of the present invention are 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 folds, oral drills, perioral folds, crow's feet, skin depressions, scars, temples, subdermal supports of eyebrows, cheek and cheek fat pads, lacrimal gutter, nose, lips, cheeks, periorbital area, suborbital area , for the treatment of facial asymmetry, the mandible and the chin. 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 1-1: Preparation of Merck hydrogel (1)

About 200 kDa of hyaluronic acid was dissolved in water to prepare 5, 15, and 30 w/v% aqueous solutions, and electron beams were applied to the prepared aqueous hyaluronic acid solution using a direct current electron-beam accelerator. Bulk hydrogels (Merckgels) of Examples 1-1 to 3-3 were prepared by inducing crosslinking of hyaluronic acid by irradiating at the dose 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 1-1: Measurement of the viscosity (viscosity) of Merck hydrogel (1)

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. The closer the electron beam dose to 3 kGy, the higher the concentration of hyaluronic acid, the better the crosslinking. It can be seen that the viscosity is high. 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 crosslinking does not occur sufficiently, and if it is too high, the hyaluronic acid chain is cut and decomposition occurs. However, it goes without saying that the present invention is not limited to any theory.

On the other hand, in the case of Examples 2-3 and 3-3, it was confirmed that Merck Gel did not have a uniform viscosity but had a partially different viscosity, 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.

Preparation 1-2: Preparation of bulk hydrogel (2)

A bulk hydrogel was prepared in the same manner as in Preparation Example 1-1, but the electron beam irradiation time was changed as shown in Table 3 below.

investigation time 10 w/v% 15 w/v% 3 kGy, 10 s Example 4-1 Example 5-1 3 kGy, 1 min Example 4-2 Example 5-2 (= Example 2-2) 3 kGy, 10 min Example 4-3 Example 5-3

Experimental Example 1-2: Viscosity measurement of bulk hydrogel (2)

The viscosity of Examples 4-1 to 5-3 was measured in the same manner as in Experimental Example 1-1, and the results are shown in Table 4 below.

Viscosity (Pa s) Example 4-1 33 Example 5-1 205 Example 4-2 679 Example 5-2 (= Example 2-2) 882 Example 4-3 48 Example 5-3 186

It can be seen that the closer the electron beam irradiation time is to 1 minute, the better the crosslinking and the higher the viscosity. This is presumably because if the irradiation time is too short or too long, crosslinking does not occur sufficiently or the decomposition of hyaluronic acid occurs. However, it goes without saying that the present invention is not limited to any theory.

Preparation Example 2: Preparation of Hydrogel Containing Micro Particles

Among the above examples, the Merck gel of Example 2-2, which showed the most ideal viscosity (250 ~ 2,500 Pa·s) as a Merck gel for pulverization into micro particles, was compressed to remove air bubbles, and a homogenizer (Ika) was used. A hydrogel having micro-sized particles was prepared by pulverizing at the same speed and time as in Table 5.

time / speed 1,000 rpm 5,000 rpm 15 seconds Example 7-1 Example 8-1 2 minutes Example 7-2 Example 8-2 10 minutes Example 7-3 Example 8-3

Experimental Example 2: Diameter and Viscosity Measurement of Microparticulate Hydrogels

The particle size and viscosity of the hydrogels of Examples 7-1 to 8-3 were measured, and the particle size was confirmed by dynamic light scattering (DLS).

1 is a result of confirming the particle size of the hydrogel by DLS, (A) to (D) show Examples 7-2, 8-2, 7-1 and 8-3, respectively. Examples 8-1 and 7-3 were difficult to obtain a clear peak. Table 6 below shows the results of measuring the viscosity.

Viscosity (Pa s) Example 7-1 144 Example 8-1 95 Example 7-2 61 Example 8-2 23 Example 7-3 14 Example 8-3 3

As shown in Figure 1 and Table 6, if the grinding time is too short or long, it can be seen that the average diameter of the particles does not deviate from 50 µm to 700 µm or is not uniform, and thus does not have an appropriate viscosity (15 to 100 Pa s) , when the speed of the homogenizer is also out of the range of 1,000 to 5,000 rpm, it can be expected that it will not have desirable particle or viscosity characteristics.

Preparation Example 3: Preparation of a hydrogel by gradually changing the dose rate (Examples 9-1, 9-2, 9-3, 9-4)

About 200 kDa hyaluronic acid was dissolved in water to prepare a 15 w/v% aqueous solution, and an electron beam was applied to the prepared hyaluronic acid aqueous solution using an electron beam accelerator in the same manner as in Tables 7 and 8 below for a total of 1 minute. By irradiation, crosslinking of hyaluronic acid was induced and Merck gel was prepared.

section ~5 seconds ~12 seconds ~17 seconds ~24 seconds ~29 seconds ~36 seconds ~41 seconds ~48 seconds ~53 seconds ~60 seconds Example 9-1 (dose rate, Gy/s) 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 Example 9-2 (dose rate, Gy/s) 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40

section ~7 seconds ~12 seconds ~19 seconds ~24 seconds ~31 seconds ~36 seconds ~43 seconds ~48 seconds ~55 seconds ~60 seconds Example 9-3 (dose rate, Gy/s) 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 30
↓
50 50
↓
30 Example 9-4 (dose rate, Gy/s) 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40 40
↓
30 30
↓
40

Experimental Example 3: Measurement of Viscosity of Bulk Hydrogel (3)

Viscosity at 1 Hz of Examples 9-1 to 9-4 was measured with an Anton-Paar MCR 302 rheometer, and the results are shown in Table 9 below.

Viscosity (Pa s) Example 9-1 947 Example 9-2 799 Example 9-3 840 Example 9-4 901

In the case of Examples 9-2 and 9-3, it can be confirmed that the viscosity improvement effect through the dose control is insignificant because the viscosity is approximately equal to or slightly lower than that of Example 2-2 in which the dose is not gradually controlled.

On the other hand, in the case of Examples 9-1 and 9-4, it can be confirmed that there is an effect of improving the viscosity as the dose is gradually controlled, specifically, the section for increasing the dose is performed for 5 seconds and the section for reducing the dose is performed for 7 seconds. .

Preparation Example 4: Preparation of a hydrogel by adding low molecular weight hyaluronic acid (Examples 10-1, 10-2)

After adding an aqueous solution of about 50 kDa low molecular weight hyaluronic acid to the hydrogel of Example 2-2 prepared in the same manner as in Preparation Example 1-1 so that the low molecular weight hyaluronic acid was 10 wt% compared to the hydrogel, 3 A bulk hydrogel was prepared by irradiating an electron beam with a dose of kGy for 10 seconds (Example 10-1), and pulverized in the same manner as in Preparation Example 2 (2 minutes, 1000 rpm) to prepare a micro-particled hydrogel ( Example 10-2).

Preparation Example 5: Preparation of a hydrogel by adding a biocompatible crosslinking agent (Examples 11-1, 11-2, 11-3, 11-4)

When preparing the bulk hydrogel as in Example 2-2, the biocompatible crosslinking agent Genipin (Sigma-Aldrich) and its derivative represented by the following Chemical Formula 1 were added to an aqueous solution of hyaluronic acid by 0.5 wt. % and then irradiated with an electron beam, maintained at 36 ° C. and stirred for 12 hours to prepare a bulk hydrogel (Examples 11-1 and 11-2), and pulverized in the same manner as in Preparation Example 2 (2 min, 1000 rpm) Micro-particulated hydrogels were prepared (Examples 11-3 and 11-4). The genipine derivative of Formula 1 below was prepared using a known synthesis method. (J. Luo, et al., ChemMedChem (2012) 1661-8)

[Formula 1]

Preparation Example 6: Re-crosslinking of a hydrogel with a small particle diameter (Example 12)

A hydrogel having nano-sized particles was obtained by irradiating 100 kGy electron beam to 5 w/v% of a 10 kDa low molecular weight hyaluronic acid aqueous solution, which was prepared in Example 8-3 (particle size 24 μm) in Preparation Example 2 above. After adding to the hydrogel of 10 wt%, a micro-particled hydrogel was prepared by irradiating an electron beam with a dose of 15 kGy for 2 minutes again. (Example 12)

Experimental Example 4: Diameter and Viscosity Measurement of Hydrogels

The particle size and viscosity of the hydrogels of Examples 10-1 to 12 were measured, and the results are shown in FIG. 2 and Table 10 below.

2 is a result of confirming the particle size of the hydrogel by DLS, (E) to (G) show Examples 10-2, 11-3, and 11-4, respectively.

Viscosity (Pa s) Example 10-1 1290 Example 11-1 1378 Example 11-2 2213 Example 12 476

As shown in Table 10, the bulk hydrogels of Example 10-1, Example 11-1 and Example 11-2 exhibited high viscosity, indicating that they were more bulky, and the hydrogel of Example 12 was micro It can be seen that the viscosity increased to the gel level.

In addition, as shown in FIG. 2 , it can be seen that the microgels prepared by pulverizing the high-viscosity gels of Examples 10-1, 11-1, and 11-2 have more uniform particle sizes.

In addition, electron beam crosslinking was attempted in the same manner as in Example 2-2 using carboxymethyldextran, cyclodextrin, and glycogen instead of hyaluronic acid, but gelation was not completely achieved.

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 using Merck gel and the same.

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 an electron beam to form a hydrogel. 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) Post process

In some embodiments, the cross-linked hydrogel may be subjected to a post-treatment process.

For example, the post-treatment process may exemplify a defoaming process of removing bubbles inside the hydrogel in a bulk state. As another example, the post-treatment process may exemplify a pulverization process of pulverizing the hydrogel in a bulk state. For another example, the post-treatment process may further include a known addition process or a sterilization process.

(B) hydrogel

The hydrogel prepared according to the present invention may have a bulk form. That is, it may be a viscoelastic material that rheologically behaves according to the shape of the reactor or the shape of the receiver. In other words, the hydrogel according to the present embodiment may be in a bulk phase rather than a particle phase.

The viscosity of the bulk-phase hydrogel may be measured at 20° C. and 1 Hz with a rheometer. For example, the viscosity of the hydrogel in the bulk phase may be about 1.0 Pa s, or about 2.0 Pa 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. 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 It may be 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 11 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 2.0 0.1 3.0 60~ 40 Preparation 2 hyaluronic acid 2.0 1.0 3.0 60~ 40 Preparation 3 hyaluronic acid 2.0 8.0 3.0 60~ 40 Preparation 4 hyaluronic acid 3.0 15.0 3.0 60~ 40 Preparation 5 hyaluronic acid 3.0 20.0 3.0 60~ 40 Preparation 6 hyaluronic acid 2.0 0.1 9.0 60~ 40 Preparation 7 hyaluronic acid 2.0 1.0 9.0 60~ 40 Preparation 8 hyaluronic acid 2.0 8.0 9.0 60~ 40 Preparation 9 hyaluronic acid 3.0 15.0 9.0 60~ 40 Preparation 10 hyaluronic acid 3.0 20.0 9.0 60~ 40 Preparation 11 hyaluronic acid 2.0 0.1 15.0 150~ 45 Preparation 12 hyaluronic acid 2.0 1.0 15.0 150~ 45 Preparation 13 hyaluronic acid 2.0 8.0 15.0 150~ 45 Preparation 14 hyaluronic acid 3.0 15.0 15.0 150~ 45 Preparation 15 hyaluronic acid 3.0 20.0 15.0 150~ 45 Preparation 16 hyaluronic acid 2.0 0.1 25.0 150~ 45 Preparation 17 hyaluronic acid 2.0 1.0 25.0 150~ 45 Preparation 18 hyaluronic acid 2.0 8.0 25.0 150~ 45 Preparation 19 hyaluronic acid 3.0 15.0 25.0 150~ 45 Preparation 20 hyaluronic acid 3.0 20.0 25.0 150~ 45 Preparation 21 hyaluronic acid 10.0 0.1 85.0 40~ 40 Preparation 22 hyaluronic acid 10.0 1.0 85.0 40~ 40 Preparation 23 hyaluronic acid 10.0 8.0 85.0 40~ 40 Preparation 24 hyaluronic acid 10.0 15.0 85.0 40~ 40 Preparation 25 hyaluronic acid 10.0 20.0 85.0 40~ 40

In addition, according to the formulation shown in Table 12 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 8.0 0.1 1.0 15~ 35 Preparation 27 Dextran 8.0 1.0 1.0 15~ 35 Preparation 28 Dextran 8.0 11.0 1.0 15~ 35 Preparation 29 Dextran 8.0 18.0 1.0 180~ 35 Preparation 30 Dextran 8.0 25.0 1.0 210~ 35 Preparation 31 Dextran 8.0 0.1 5.0 15~ 35 Preparation 32 Dextran 8.0 1.0 5.0 15~ 35 Preparation 33 Dextran 8.0 11.0 5.0 15~ 35 Preparation 34 Dextran 8.0 18.0 5.0 180~ 35 Preparation 35 Dextran 8.0 25.0 5.0 210~ 35 Preparation 36 Dextran 12.0 0.1 15.0 15~ 35 Preparation 37 Dextran 12.0 1.0 15.0 15~ 35 Preparation 38 Dextran 12.0 11.0 15.0 15~ 35 Preparation 39 Dextran 12.0 18.0 15.0 180~ 35 Preparation 40 Dextran 12.0 25.0 15.0 210~ 35 Preparation 41 Dextran 12.0 0.1 30.0 15~ 35 Preparation 42 Dextran 12.0 1.0 30.0 15~ 35 Preparation 43 Dextran 12.0 11.0 30.0 15~ 35 Preparation 44 Dextran 12.0 18.0 30.0 180~ 35 Preparation 45 Dextran 12.0 25.0 30.0 210~ 35

In addition, according to the formulation shown in Table 13 below, an aqueous solution of polysaccharide 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 1.0 0.1 3.0 30~ 39 Preparation 47 fucoidan 1.0 4.0 3.0 30~ 39 Preparation 48 fucoidan 1.0 10.0 3.0 30~ 39 Preparation 49 fucoidan 1.0 15.0 3.0 30~ 39 Preparation 50 fucoidan 1.0 20.0 3.0 30~ 39 Preparation 51 fucoidan 1.0 0.1 10.0 30~ 39 Preparation 52 fucoidan 1.0 4.0 10.0 30~ 39 Preparation 53 fucoidan 1.0 10.0 10.0 30~ 39 Preparation 54 fucoidan 1.0 15.0 10.0 30~ 39 Preparation 55 fucoidan 1.0 20.0 10.0 30~ 39 Preparation 56 fucoidan 4.0 0.1 50.0 30~ 39 Preparation 57 fucoidan 4.0 4.0 50.0 30~ 39 Preparation 58 fucoidan 4.0 10.0 50.0 30~ 39 Preparation 59 fucoidan 4.0 15.0 50.0 30~ 39 Preparation 60 fucoidan 4.0 20.0 50.0 30~ 39

In addition, according to the formulation shown in Table 14 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 Carboxymethyldextran 5.0 1.0 1.0 20~ 44 Preparation 62 Carboxymethyldextran 5.0 4.0 1.0 20~ 44 Preparation 63 Carboxymethyldextran 5.0 8.0 1.0 20~ 44 Preparation 64 Carboxymethyldextran 5.0 10.0 1.0 20~ 44 Preparation 65 Carboxymethyldextran 10.0 15.0 1.0 20~ 41 Preparation 66 Carboxymethyldextran 5.0 1.0 4.0 20~ 44 Preparation 67 Carboxymethyldextran 5.0 4.0 4.0 20~ 44 Preparation 68 Carboxymethyldextran 5.0 8.0 4.0 20~ 44 Preparation 69 Carboxymethyldextran 5.0 10.0 4.0 20~ 44 Preparation 70 Carboxymethyldextran 10.0 15.0 4.0 20~ 41

In addition, according to the formulation shown in Table 15 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 71 alginate 5.0 2.0 3.0 120~ 43 Preparation 72 alginate 5.0 5.0 4.0 120~ 43 Preparation 73 alginate 5.0 10.0 5.0 120~ 44 Preparation 74 alginate 5.0 12.0 6.0 120~ 44 Preparation 75 alginate 5.0 20.0 10.0 150~ 45 Preparation 76 chitosan 2.5 2.0 4.0 220~ 44 Preparation 77 chitosan 2.5 5.0 5.0 220~ 44 Preparation 78 chitosan 2.5 10.0 6.0 220~ 45 Preparation 79 chitosan 2.5 12.0 7.0 220~ 46 Preparation 80 chitosan 2.5 20.0 8.5 250~ 46

In addition, according to the formulation shown in Table 16 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 81 gelatin 5.0 3.0 5.0 40~ 45 Preparation 82 gelatin 5.0 3.0 10.0 40~ 45 Preparation 83 gelatin 5.0 5.0 10.0 40~ 45 Preparation 84 met 10.0 4.0 3.0 40~ 30 Preparation 85 met 10.0 8.0 3.0 40~ 30 Preparation 86 met 10.0 10.0 9.5 40~ 30 Preparation 87 β-cyclodextrin 7.0 4.0 10.0 40~ 38 Preparation 88 β-cyclodextrin 7.0 4.0 15.0 40~ 38 Preparation 89 β-cyclodextrin 7.0 4.0 25.0 40~ 38

According to the present invention, Merck gel can be formed only by electron beam crosslinking, beyond simply increasing the particle size from nano size to micro size. In addition, since Merck Gel has excellent viscosity and elasticity, it is easy to control the particle size through pulverization, etc., and has the effect that it can be used in various fields.

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)

Preparing a bulk hydrogel (hydrogel) by irradiating an electron-beam to an aqueous solution of hyaluronic acid at a dose of 0.5 to 5 kGy for 30 seconds to 5 minutes to crosslink the hyaluronic acid; and
A method for producing a Merck gel for filler treatment, comprising the step of removing air bubbles inside the bulk hydrogel.

Images