KR102626896B1 - Collagen Hydrogel comprising Crosslinked Collagen and Method for Manufacturing thereof - Google Patents

Abstract

The present invention relates to a collagen hydrogel in which the cross-linking of the collagen matrix is promoted and a method for manufacturing the same. More specifically, the present invention relates to a collagen hydrogel in which the cross-linking of the collagen matrix is promoted and a method of manufacturing the same. More specifically, the cross-linking degree of the collagen matrix is promoted by using riboflavin phosphate (RFP), a biocompatible and non-toxic natural photoinitiator, and blue light. It relates to a collagen hydrogel that promotes cross-linking of the collagen matrix, which can suppress the hardening or stiffening of the collagen matrix, and a method of manufacturing the same.

Description

Collagen hydrogel promoting crosslinking of collagen matrix and method for manufacturing same {Collagen Hydrogel comprising Crosslinked Collagen and Method for Manufacturing thereof}

The present invention relates to a collagen hydrogel in which the cross-linking of the collagen matrix is promoted and a method for manufacturing the same. More specifically, the present invention relates to a collagen hydrogel in which the cross-linking of the collagen matrix is promoted and a method of manufacturing the same. More specifically, the cross-linking degree of the collagen matrix is promoted by using riboflavin phosphate (RFP), a biocompatible and non-toxic natural photoinitiator, and blue light. It relates to a collagen hydrogel that promotes cross-linking of the collagen matrix, which can suppress the hardening or stiffening of the collagen matrix, and a method of manufacturing the same.

In modern society, as cutting-edge science and technology develop, bio, medical, and cosmetic technologies also develop. As life expectancy and quality of life have improved, people have become a society that pursues abundance, and interest in skin care and anti-aging has also increased. Aging of the skin results in loss of elasticity due to a decrease or destruction of collagen in skin tissue and an increase in wrinkles and pigmentation as the skin layer becomes thinner. Therefore, several treatment methods such as retinol, chemical peeling, and laser have been used to promote collagen biosynthesis and restore aged collagen matrix within the skin layer. However, these methods carry a risk of side effects such as erythema, pain, infection, burns, pigmentation, and scarring.

Various procedures and beauty devices to prevent or restore skin aging continue to appear. Currently, among the beauty devices that have appeared for skin care, the device that uses light is the LED mask. LED masks are cheaper, safer, and easier to use than existing laser devices, so they are widely used in home cosmetics and medical devices. LED masks determine the stimulation area of the skin layer depending on the wavelength and are used for acne treatment in the epidermis and dermis, inflammation healing in the dermis, and skin regeneration. However, in the case of LED masks, since they only use light, the stimulation efficiency is reduced and the time in contact with the skin increases, and due to the nature of the semiconductor device that emits light by electric current, it is exposed to harmful electromagnetic waves and affects the skin.

Collagen is known to be the most abundant protein in animals and humans. Because collagen accounts for a large percentage of tissue components, it is widely used as a biomaterial for tissue engineering. However, because collagen has weak mechanical properties and rapid decomposition, it is difficult to handle clinically without modification. To overcome these limitations, various chemical collagen cross-links have been developed, among which methods using glutaraldehyde and formaldehyde are representative. However, these chemical methods are still vulnerable to cytotoxicity and have the side effect of causing tissue calcification, so research to secure these areas is urgently needed.

The present invention uses riboflavin phosphate (RFP), a biocompatible and non-toxic natural photoinitiator, and blue light to promote the degree of cross-linking of the collagen matrix and to suppress the hardening or stiffening of the collagen matrix. The purpose is to provide collagen hydrogel and its manufacturing method.

In order to solve the above problems, in one embodiment of the present invention, as a method for producing collagen hydrogel, a collagen hydrogel solution is prepared by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution. steps; Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with light having a wavelength range of 365 to 460 nm for 1 to 60 minutes; and incubating the preliminary collagen hydrogel at a temperature of 35 to 40° C. for 2 to 4 hours to form a collagen hydrogel.

In some embodiments of the present invention, preparing the collagen hydrogel solution includes preparing a neutralizing solution of pH 4 to 9; Preparing riboflavin phosphate (RFP) with a concentration of 5 to 15 mg/ml by diluting in PBS; Preparing a mixture by mixing the neutralization solution and collagen at a volume ratio of 4:1 to 1:4; And it may include preparing a collagen hydrogel solution by mixing 0.001 to 0.1% by weight of riboflavin phosphate (RFP) with the mixture based on the total weight of the mixture.

In some embodiments of the present invention, the collagen may include one of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen.

In order to solve the above problems, in one embodiment of the present invention, as a collagen hydrogel, the steps of preparing a collagen hydrogel solution by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution; Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with light having a wavelength range of 365 to 460 nm for 1 to 60 minutes; and incubating the preliminary collagen hydrogel at a temperature of 35 to 40° C. for 2 to 4 hours to form a collagen hydrogel.

In order to solve the above problems, in one embodiment of the present invention, a method of promoting cross-linking of a collagen matrix includes the steps of applying riboflavin phosphate (RFP) to a target containing collagen; And irradiating light having a wavelength range of 365 to 460 nm while the riboflavin phosphate (RFP) is applied to the object containing the collagen. do.

In order to solve the above problems, in one embodiment of the present invention, a cosmetic set product that promotes the creation of cross-linking of the collagen matrix is provided, and the first cosmetic product is a lotion, skin, wrinkle improvement cream, eye cream, or face oil. A cosmetic comprising a composition and a second cosmetic composition mixed with 0.001 to 0.1% by weight of riboflavin phosphate (RFP) based on the total weight of the first cosmetic composition; and a light irradiation device capable of irradiating light having a wavelength range of 365 to 460 nm to the object to which the cosmetic is applied.

According to one embodiment of the present invention, as the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light, the effect of promoting the degree of cross-linking of collagen hydrogel or objects containing collagen can be exerted.

According to one embodiment of the present invention, as the degree of cross-linking of the collagen matrix is promoted, the phenomenon of hardening or stiffening of the collagen matrix is suppressed, which has the effect of improving the moisture content of collagen hydrogel or an object containing collagen. there is.

According to one embodiment of the present invention, by irradiating blue light when cross-linking the collagen matrix, the amount of electromagnetic waves exposed to collagen hydrogel or objects containing collagen can be reduced, and side effects such as burns are minimized, making it safer. This can have the effect of improving the degree of cross-linking.

According to one embodiment of the present invention, as the irradiation time of blue light increases, cross-linking of the collagen matrix is promoted to form a hydrogel, so the collagen hydrogel exhibits elastic behavior, which has the effect of improving elasticity. .

According to one embodiment of the present invention, the effect of improving the irradiation effect of a light irradiation device can be achieved by combining riboflavin phosphate (RFP) and blue light.

According to one embodiment of the present invention, a riboflavin phosphate (RFP) solution diluted to a preset concentration range is applied to an object containing collagen, and blue light is shined on the object containing collagen to which the riboflavin phosphate (RFP) solution has been applied. By irradiating within a set time range, the Young's modulus and tensile strength of an object containing collagen can be improved, thereby improving elasticity.

Figure 1 schematically shows a method for producing collagen hydrogel according to an embodiment of the present invention.
Figure 2 schematically shows the collagen matrix inside the collagen hydrogel according to an embodiment of the present invention.
Figure 3 schematically shows the detailed steps of preparing a collagen hydrogel solution according to an embodiment of the present invention.
Figure 4 schematically shows the degree of gelation of the pre-collagen hydrogel according to an embodiment of the present invention.
Figure 5 schematically shows FT-IR analysis data of collagen hydrogel according to an embodiment of the present invention.
Figure 6 schematically shows rheological analysis data of collagen hydrogel according to an embodiment of the present invention.
Figure 7 schematically shows a cross-sectional SEM photograph of a collagen hydrogel according to an embodiment of the present invention.
Figure 8 schematically shows a cross-linking acceleration experiment according to an embodiment of the present invention.
Figure 9 schematically shows physical property data according to the concentration of riboflavin phosphate (RFP), among the results of a crosslinking acceleration experiment according to an embodiment of the present invention.
Figure 10 schematically shows physical property data according to the irradiation time of blue light, among the results of a crosslinking acceleration experiment in one embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments and/or aspects are now disclosed with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth to facilitate a general understanding of one or more aspects. However, it will also be appreciated by those skilled in the art that this aspect(s) may be practiced without these specific details. The following description and accompanying drawings set forth in detail certain example aspects of one or more aspects. However, these aspects are illustrative and some of the various methods in the principles of the various aspects may be utilized, and the written description is intended to encompass all such aspects and their equivalents.

Additionally, the term “or” is intended to mean an inclusive “or” and not an exclusive “or.” That is, unless otherwise specified or clear from context, “X utilizes A or B” is intended to mean one of the natural implicit substitutions. That is, either X uses A; X uses B; Or, if X uses both A and B, “X uses A or B” can apply to either of these cases. Additionally, the term “and/or” as used herein should be understood to refer to and include all possible combinations of one or more of the related listed items.

Additionally, the terms "comprise" and/or "comprising" mean that the feature and/or element is present, but exclude the presence or addition of one or more other features, elements and/or groups thereof. It should be understood as not doing so.

Additionally, in this specification, it will be understood that singular expressions such as “unless” and “above” include plural expressions unless otherwise clearly indicated.

Additionally, terms including ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, a first component may be named a second component, and similarly, the second component may also be named a first component without departing from the scope of the present invention. The term and/or includes any of a plurality of related stated items or a combination of a plurality of related stated items.

In addition, in the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, are generally understood by those skilled in the art to which the present invention pertains. It has the same meaning as Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the embodiments of the present invention, have an ideal or excessively formal meaning. It is not interpreted as

Conventionally, methods such as retinol, chemical peeling, and laser were used to restore the collagen matrix. However, this method can cause side effects such as erythema, pain, and scarring, so recently, a method that induces cross-linking of the collagen matrix has been used.

However, conventional cross-linking is induced using a cross-linking agent such as Palladium bacterial pheophorbide and an LED mask, so there is a problem that the collagen matrix may harden or become rigid.

To solve this problem, in the present invention, riboflavin phosphate (RFP), a natural photoinitiator, and blue light were used to promote photo-crosslinking of the collagen matrix.

In this case, the degree of cross-linking of the collagen matrix can be promoted to improve moisture content and elasticity, and the time exposed to electromagnetic waves can be reduced, thereby minimizing side effects and restoring the collagen matrix more safely. Hereinafter, the collagen hydrogel of the present invention, in which cross-linking is promoted in a special manner, will be described in detail.

Figure 1 schematically shows a method for producing collagen hydrogel according to an embodiment of the present invention.

A method for producing a collagen hydrogel according to an embodiment of the present invention, comprising the steps of preparing a collagen hydrogel solution by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution (S100); Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with light having a wavelength range of 365 to 460 nm for 1 to 60 minutes (S200); and incubating the preliminary collagen hydrogel at a temperature of 35 to 40° C. for 2 to 4 hours to form a collagen hydrogel (S300).

In the present invention, through the above steps, a collagen hydrogel in which the crosslinking degree of the collagen matrix is promoted can be produced.

As the degree of cross-linking of the collagen matrix is promoted by this manufacturing method, hardening or stiffening of the collagen matrix is suppressed, which has the effect of improving the water content of the collagen hydrogel.

Preferably, the collagen hydrogel includes the steps of preparing a collagen hydrogel solution by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution (S100); Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with light having a wavelength range of 365 to 460 nm for 1 to 60 minutes (S200); And incubating the preliminary collagen hydrogel at a temperature of 35 to 40 ° C. for 2 to 4 hours to form a collagen hydrogel (S300).

As shown in Figure 1, in step S100, a collagen hydrogel solution can be prepared by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution.

The collagen according to an embodiment of the present invention may include one of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. The neutralization solution may include a neutralization solution with a pH of 4 to 9. The riboflavin phosphate corresponds to a vitamin B derivative (Riboflavin phosphate (RFP)) and may correspond to a relatively non-toxic natural photoinitiator.

In step S200, a preliminary collagen hydrogel can be formed by irradiating the collagen hydrogel solution with light having a wavelength range of 365 to 460 nm for 1 to 60 minutes. In one embodiment of the present invention, the light having a wavelength range of 365 to 460 nm may include blue light. The blue light has relatively high stability compared to the UV generated from conventional LED masks. In the case of light having a wavelength range other than the above wavelength range, it is not suitable for promoting cross-linking of the collagen matrix, so it is preferable to use light having the above wavelength range.

의 온도에서 2 내지 4시간 동안 인큐베이션하여 콜라겐하이드로젤을 형성할 수 있다. 바람직하게는, 상기 단계 S300에서는 상기 예비콜라겐하이드로젤을 37

의 온도에서 3시간 동안 인큐베이션하여 콜라겐하이드로젤을 형성할 수 있다.In step S300, the pre-collagen hydrogel is 35 to 40

Collagen hydrogel can be formed by incubating at a temperature for 2 to 4 hours. Preferably, in step S300, the pre-collagen hydrogel is 37

Collagen hydrogel can be formed by incubating for 3 hours at a temperature of .

As described above, a photoinitiator and light are required for photocrosslinking of a collagen matrix. In the present invention, riboflavin phosphate (RFP) is used as a photoinitiator, and light with a wavelength range of 365 to 460 nm, that is, blue light, is used as light. did.

In this way, the collagen hydrogel according to an embodiment of the present invention can be cross-linked using a combination of riboflavin phosphate (RFP) and blue light. Preferably, as the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light, the effect of promoting the degree of cross-linking of the collagen hydrogel can be exerted. In addition, by irradiating blue light during crosslinking of the collagen matrix, it can have the effect of reducing the amount of electromagnetic waves exposed to the collagen hydrogel.

Figure 2 schematically shows the collagen matrix inside the collagen hydrogel according to an embodiment of the present invention.

In one embodiment of the present invention, the collagen hydrogel contains riboflavin phosphate (RFP) and can be crosslinked by light with a wavelength of 458 nm. The cross-linked collagen hydrogel can exhibit a planar porous structure as shown in Figure 2, and as the irradiation time of the light increases in the state in which riboflavin phosphate (RFP) is added, the planar porous structure decreases. It can exhibit a dense porous structure. At this time, it can be seen that the spacing and size between the network structures can be reduced together, and the degree of cross-linking increases.

In other words, as the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light, the effect of promoting the degree of cross-linking of the collagen hydrogel can be exerted.

Figure 3 schematically shows the detailed steps of preparing a collagen hydrogel solution according to an embodiment of the present invention.

Preparing the collagen hydrogel solution according to an embodiment of the present invention (S100) includes preparing a neutralizing solution of pH 4 to 9 (S110); Preparing riboflavin phosphate (RFP) with a concentration of 5 to 15 mg/ml by diluting it in PBS (S120); Preparing a mixture by mixing the neutralization solution and collagen at a volume ratio of 4:1 to 1:4 (S130); And it may include preparing a collagen hydrogel solution by mixing 0.001 to 0.1% by weight of riboflavin phosphate (RFP) with the mixture based on the total weight of the mixture (S140).

In step S110, a neutralization solution with a pH of 4 to 9 can be prepared. In one embodiment of the present invention, the neutralization solution may be prepared by mixing 1300 to 1500 uL of DI water, 400 to 600 uL of 10X PBS, and 60 to 80 uL of NaOH. Preferably, the neutralization solution may be a neutralization solution prepared by mixing 1400 to 1450 uL of DI water, 450 to 550 uL of 10X PBS, and 70 to 80 uL of NaOH, and more preferably, the neutralization solution is DI water 1425. It may be a neutralization solution prepared by mixing uL, 500 uL of 10X PBS, and 75 uL of NaOH. However, the neutralization solution is not limited to this and may include all neutralization solutions corresponding to the acidic concentration range of 4 to 9 pH.

In step S120, riboflavin phosphate (RFP) having a concentration of 5 to 15 mg/ml can be prepared by diluting in PBS. Preferably, in step S120, riboflavin phosphate (RFP) having a concentration of 8 to 12 mg/ml can be prepared by diluting in PBS, and more preferably, in step S120, riboflavin phosphate (RFP) can be prepared by diluting in PBS to have a concentration of 10 mg/ml. Riboflavin phosphate (RFP) can be prepared with any concentration.

In step S130, a mixture can be prepared by mixing the neutralization solution and collagen at a volume ratio of 4:1 to 1:4. In one embodiment of the present invention, in step S130, a mixture may be prepared by mixing the neutralization solution and the collagen at a volume ratio of 3:2. At this time, it is preferable that the neutralization solution and the collagen are mixed in a tube or vial.

In step S140, a collagen hydrogel solution can be prepared by mixing 0.001 to 0.1% by weight of riboflavin phosphate (RFP) with the mixture based on the total weight of the mixture. When the riboflavin phosphate (RFP) is added to the mixture in an amount exceeding 0.1% by weight, the light irradiated to the mixture may only penetrate the surface of the mixture, so crosslinking will not be promoted inside the mixture. You can.

In one embodiment of the present invention, in step S140, the collagen hydrogel solution can be prepared by mixing 0.01% by weight of riboflavin phosphate (RFP) based on the total weight of the mixture.

That is, the collagen hydrogel solution prepared through the above steps contains riboflavin phosphate (RFP), a natural photoinitiator, as a photoinitiator, and can suppress the phenomenon of hardening or stiffening of the collagen matrix after crosslinking compared to technology using a conventional crosslinking agent. there is.

Preferably, the collagen hydrogel solution according to an embodiment of the present invention can suppress hardening or stiffening of the collagen matrix, thereby improving the water content of the collagen hydrogel.

Figure 4 schematically shows the degree of gelation of the pre-collagen hydrogel according to an embodiment of the present invention.

As described above, the preliminary collagen hydrogel may be formed by irradiating light having a wavelength range of 365 to 460 nm to the collagen hydrogel solution, and the light may include blue light.

The collagen hydrogel solution is prepared by adding collagen to the neutralization solution at a preset volume ratio as shown in FIG. 4(a) to prepare a mixture, and then adding the riboflavin phosphate (RFP) prepared in the mixture as shown in FIG. 4(b). It can be prepared by adding it in a preset weight %. When the prepared collagen hydrogel solution is irradiated with blue light having the above-mentioned wavelength range, crosslinking is promoted and gelation can occur, as shown in FIG. 4(c). In one embodiment of the present invention, the collagen hydrogel solution can be irradiated with blue light using a dental LED curing lamp.

Below, the chemical structure, rheology, and cross-section were analyzed to confirm the characteristics of the collagen hydrogel according to an embodiment of the present invention.

Figure 5 schematically shows FT-IR analysis data of collagen hydrogel according to an embodiment of the present invention.

FT-IR analysis was performed to confirm the uniqueness of the change in functional group composition of the collagen hydrogel during the cross-linking reaction, and was performed within the wavelength range of 600 to 1800 cm ^-1 .

In order to perform FT-IR analysis, the collagen hydrogel according to an embodiment of the present invention was prepared by freeze-drying for 24 hours. At this time, the collagen hydrogel was prepared in a physically crosslinked state (“Physical Crosslinking” in Figure 5) and a photocrosslinked state (“Chemical Crosslinking” in Figure 5).

As a result of FT-IR analysis, as shown in Figure 5, the photo-crosslinked collagen hydrogel had amide I and II at a wavelength of 1640 cm ^-1 and 1560 cm ^-1 , respectively, compared to the physically cross-linked collagen hydrogel. Absorption and the intensity of the absorption peak representing the C=N bond were relatively increased, and the peak representing the amine C-N bond was relatively decreased at a wavelength of 1240 cm ^-1 . This can be determined that the amine group was consumed to form a covalent bond during photo-crosslinking.

In addition, the photo-crosslinked collagen hydrogel had a relatively increased peak at a wavelength of 1170 cm ^-1 , indicating the CO bond of the ester group. It can be judged that the high-energy oxygen atom induces a covalent bond between the proline and histidine residues of adjacent collagen and forms an additional CO ester bond.

That is, as shown in Figure 5, it can be confirmed that the carbonyl-amine reaction and the proline-histidine reaction proceed in the collagen hydrogel according to an embodiment of the present invention when photo-crosslinking proceeds.

Figure 6 schematically shows rheological analysis data of collagen hydrogel according to an embodiment of the present invention.

Rheological analysis is to confirm the change in the physical properties of the collagen hydrogel according to the irradiation time of blue light and the functional group composition. During the experiment, the temperature was maintained at 37 ° C., the strain was fixed at 1%, and the The storage modulus (“G'” in Figure 6) and loss modulus (“G”” in Figure 6) were measured at a range of frequencies.

In order to perform rheological analysis, the storage modulus and loss modulus of the collagen hydrogel were measured using a rheometer immediately after producing the collagen hydrogel according to an embodiment of the present invention, and the physically cross-linked Photo-crosslinked collagen hydrogel samples were prepared by irradiating the collagen hydrogel samples with blue light for 1 minute, 5 minutes, 10 minutes, 20 minutes, and 30 minutes.

Figure 6(a) shows the rheology of physically cross-linked collagen hydrogel, and Figures 6(b) to 6(f) show blue light for 1 minute, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. The rheology of the photo-crosslinked collagen hydrogel is shown during irradiation.

As a result of the rheological analysis, it can be seen that the collagen hydrogel, except for the physically cross-linked collagen hydrogel in Figure 6(a), has a storage modulus higher than the loss modulus, showing viscoelastic behavior. It can be determined that all photo-crosslinked collagen hydrogel samples in Figures 6(b) to 6(f) formed hydrogels.

Meanwhile, Figure 6(g) shows a graph of the storage modulus according to the irradiation time of blue light for all collagen hydrogels in Figures 6(a) to 6(f). As shown in Figure 6(g), it can be seen that the storage modulus of the collagen hydrogel increases as the irradiation time of blue light increases. It can be judged that the collagen hydrogel exhibits elastic behavior as cross-linking of the collagen matrix is promoted by the action of riboflavin phosphate (RFP) and blue light to form a hydrogel.

Preferably, in one embodiment of the present invention, as the irradiation time of blue light increases, cross-linking of the collagen matrix is promoted to form a hydrogel, so that the collagen hydrogel exhibits elastic behavior to improve elasticity. You can.

Figure 7 schematically shows a cross-sectional SEM photograph of a collagen hydrogel according to an embodiment of the present invention.

The cross-sectional SEM photo was used to confirm the cross-section of the collagen hydrogel, and was confirmed using a scanning electron microscope (SEM).

In order to check the cross-sectional SEM image, immediately after manufacturing the collagen hydrogel according to an embodiment of the present invention, it was frozen with liquid nitrogen, the cross-section was cut and freeze-dried for 24 hours, and the cross-section was coated with Pt in a vacuum. Analyzed, the physically cross-linked collagen hydrogel sample was prepared by irradiating the sample with blue light for 1 minute, 5 minutes, 10 minutes, 20 minutes, and 30 minutes.

Figure 7(a) shows a cross-sectional SEM of a physically cross-linked collagen hydrogel, and Figures 7(b) to 7(f) show blue light for 1 minute, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. Shown is a cross-sectional SEM of the photo-crosslinked collagen hydrogel during irradiation.

As a result of cross-sectional analysis, it was confirmed that the pore size of the collagen hydrogel decreased and the density increased as the irradiation time of blue light increased.

5 to 7, the collagen hydrogel containing riboflavin phosphate (RFP), a natural photoinitiator, is photo-crosslinked by blue light, thereby causing carbonyl-amine reaction and proline-histidine reaction to proceed, forming the hydrogel. It exhibits viscoelastic behavior with a relatively high storage modulus compared to the loss modulus. As the irradiation time of blue light increases, cross-linking of the collagen matrix is promoted, the storage modulus increases, showing elastic behavior, and the size of the pores decreases, resulting in an increase in density. there is.

In this way, the collagen hydrogel according to an embodiment of the present invention can exert the effect of promoting the degree of cross-linking of the collagen hydrogel as the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light.

Meanwhile, the present invention provides a method of promoting the creation of cross-links in a collagen matrix using a combination of riboflavin phosphate (RFP) and blue light.

Preferably, the method of promoting cross-linking of the collagen matrix according to an embodiment of the present invention includes the steps of applying riboflavin phosphate (RFP) to a target containing collagen; and irradiating light having a wavelength range of 365 to 460 nm while the riboflavin phosphate (RFP) is applied to the object containing the collagen. At this time, light having a wavelength range of 365 to 460 nm may correspond to blue light.

By this method, in one embodiment of the present invention, the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light, thereby achieving the effect of promoting the degree of cross-linking of the object containing the collagen. In addition, as the degree of cross-linking of the collagen matrix is promoted, the phenomenon of hardening or stiffening of the collagen matrix is suppressed, which has the effect of improving the moisture content of the object containing collagen.

In addition, the present invention provides a cosmetic set product that promotes the creation of cross-links in the collagen matrix using a combination of riboflavin phosphate (RFP) and blue light.

Preferably, the cosmetic set product that promotes the creation of cross-linking of the collagen matrix according to an embodiment of the present invention includes a first cosmetic composition corresponding to lotion, skin, wrinkle improvement cream, eye cream, or face oil, and A cosmetic comprising a second cosmetic composition mixed with 0.001 to 0.1% by weight of riboflavin phosphate (RFP) based on the total weight of the first cosmetic composition; and a light irradiation device capable of irradiating light having a wavelength range of 365 to 460 nm to the object to which the cosmetic is applied. At this time, the light having a wavelength range of 365 to 460 nm may correspond to blue light, and the light irradiation device may include one of an LED mask and a dental LED curing lamp.

By using such a cosmetic set product, in one embodiment of the present invention, blue light is irradiated during crosslinking of the collagen matrix, thereby reducing the amount of electromagnetic waves exposed to objects containing collagen and minimizing side effects such as burns. It can be effective in improving the degree of cross-linking in a safe manner. Additionally, the combination of riboflavin phosphate (RFP) and blue light can improve the irradiation effect of the light irradiation device.

Below, a cross-linking promotion experiment was performed on the collagen-containing object using riboflavin phosphate (RFP) and blue light.

Figure 8 schematically shows a cross-linking acceleration experiment according to an embodiment of the present invention.

This experiment is to determine the effect on the degree of crosslinking of the collagen-containing object when riboflavin phosphate (RFP) is applied to the collagen-containing object and blue light is irradiated.

In this experiment, pig skin was selected as the collagen-containing object, and the pig skin was prepared by cutting it into a 5cm x 1cm rectangle as shown in FIG. 8(a). At this time, the pig skin used The Micropig® Franz cell membranes, and 12 samples were prepared by dividing them into 3 groups of 4 each.

Pig skin samples from three groups were each administered a solution containing 0.01% by weight of riboflavin phosphate (RFP), a solution containing 0.05% by weight of riboflavin phosphate (RFP), and 0.1% by weight of riboflavin phosphate (RFP). After incubating for 1 hour in the solution containing the solution as shown in Figure 8(b), the surface was gently washed using PBS.

Then, as shown in Figure 8(c), the pig skin samples of each group were exposed to light with a wavelength range of 365 to 460 nm for 1 minute, 5 minutes, 10 minutes, and 30 minutes, respectively, as shown in Figure 8(c). During the investigation.

The tensile strength of the pig skin samples prepared through this process was measured immediately after production, and the tensile strength was measured at a speed of 1 cm/min using a 5N load cell until each pig skin sample was damaged, and Young's Modulus was measured. and tensile strength were measured.

Figure 9 schematically shows physical property data according to the concentration of riboflavin phosphate (RFP), among the results of a crosslinking acceleration experiment according to an embodiment of the present invention.

Figure 9(a) shows the change in Young's modulus according to the concentration of riboflavin phosphate (RFP), and Figure 9(b) shows the change in tensile strength according to the concentration of riboflavin phosphate (RFP). For this data, the Young's modulus and tensile strength were measured by changing only the RFP concentration of a solution containing riboflavin phosphate (RFP) after fixing the light irradiation time to 5 minutes.

As a result of the analysis, compared to the sample soaked for 1 hour in a solution without riboflavin phosphate (RFP) (“PBS” in Figure 9), all samples treated with the solution (“RFP 0.01%” in Figure 9, “RFP 0.05%”) ”, and “RFP 0.1%”), it can be seen that the Young’s modulus and tensile strength have improved. In particular, it can be seen that the RFP 0.05% sample, which is a sample treated in a solution containing 0.05% by weight of riboflavin phosphate (RFP), showed the most improved results.

In one embodiment of the present invention, the method of promoting cross-linking of the collagen matrix may include applying a solution containing 0.01 to 0.1% by weight of riboflavin phosphate (RFP) to an object containing collagen. Preferably, the method of promoting cross-linking of the collagen matrix may include applying a solution containing 0.05% by weight of riboflavin phosphate (RFP) to an object containing collagen.

That is, in one embodiment of the present invention, the Young's modulus and tensile strength of the object containing collagen are increased by applying a riboflavin phosphate (RFP) solution diluted to a preset concentration range and irradiating blue light to the object containing collagen. It can have the effect of improving elasticity.

Figure 10 schematically shows physical property data according to the irradiation time of blue light, among the results of a crosslinking acceleration experiment in one embodiment of the present invention.

Figure 10(a) shows the change in Young's modulus according to the light irradiation time, and Figure 10(b) shows the change in tensile strength according to the light irradiation time. This data was obtained by fixing the concentration of riboflavin phosphate (RFP) at 0.05% by weight and then measuring Young's modulus and tensile strength by changing only the time for irradiating light to the sample.

As a result of the analysis, it was confirmed that the Young's modulus and tensile strength of the sample improved as the light irradiation time increased. However, when the irradiation time of light exceeds 10 minutes, it can be confirmed that the effect of the irradiation time is minimal.

In one embodiment of the present invention, the method of promoting the creation of cross-links in the collagen matrix includes, in a state in which the riboflavin phosphate (RFP) is applied to an object containing the collagen, having a wavelength range of 365 to 460 nm. Light can be irradiated for 1 to 30 minutes. Preferably, the method of promoting the creation of cross-linking of the collagen matrix includes applying light with a wavelength of 365 to 460 nm in a state in which the riboflavin phosphate (RFP) is applied to the object containing the collagen. You can investigate for 10 minutes.

That is, in one embodiment of the present invention, by irradiating blue light to an object containing collagen to which a riboflavin phosphate (RFP) solution is applied in a preset time range, the Young's modulus and tensile strength of the object containing collagen are improved. It can have the effect of improving elasticity.

According to one embodiment of the present invention, as the collagen matrix is cross-linked by riboflavin phosphate (RFP) and blue light, the effect of promoting the degree of cross-linking of collagen hydrogel or objects containing collagen can be exerted.

According to one embodiment of the present invention, as the degree of cross-linking of the collagen matrix is promoted, the phenomenon of hardening or stiffening of the collagen matrix is suppressed, which has the effect of improving the moisture content of collagen hydrogel or an object containing collagen. there is.

According to one embodiment of the present invention, by irradiating blue light when cross-linking the collagen matrix, the amount of electromagnetic waves exposed to collagen hydrogel or objects containing collagen can be reduced, and side effects such as burns are minimized, making it safer. This can have the effect of improving the degree of cross-linking.

According to one embodiment of the present invention, as the irradiation time of blue light increases, cross-linking of the collagen matrix is promoted to form a hydrogel, so the collagen hydrogel exhibits elastic behavior, which has the effect of improving elasticity. .

According to one embodiment of the present invention, the effect of improving the irradiation effect of a light irradiation device can be achieved by combining riboflavin phosphate (RFP) and blue light.

According to one embodiment of the present invention, a riboflavin phosphate (RFP) solution diluted to a preset concentration range is applied to an object containing collagen, and blue light is shined on the object containing collagen to which the riboflavin phosphate (RFP) solution has been applied. By irradiating within a set time range, the Young's modulus and tensile strength of an object containing collagen can be improved, thereby improving elasticity.

As described above, although the embodiments have been described with limited examples and drawings, various modifications and variations can be made by those skilled in the art from the above description. For example, the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or other components are used. Alternatively, appropriate results may be achieved even if replaced or substituted by an equivalent. Therefore, other implementations, other embodiments, and equivalents of the claims also fall within the scope of the claims described below.

Claims (6)

As a method for producing collagen hydrogel,
Preparing a collagen hydrogel solution by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution;
Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with blue light having a wavelength range of 450 to 460 nm for 1 to 60 minutes; and
Incubating the pre-collagen hydrogel at a temperature of 35 to 40° C. for 2 to 4 hours to form a collagen hydrogel;
The step of preparing the collagen hydrogel solution is,
Preparing a neutralizing solution of pH 4 to 9;
Preparing riboflavin phosphate (RFP) with a concentration of 5 to 15 mg/ml by diluting in PBS;
Preparing a mixture by mixing the neutralization solution and collagen at a volume ratio of 4:1 to 1:4; and
Preparing a collagen hydrogel solution by mixing 0.01 to 0.1% by weight of riboflavin phosphate (RFP) with the mixture based on the total weight of the mixture.
delete In claim 1,
A method of producing a collagen hydrogel, wherein the collagen includes one of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen.
As a collagen hydrogel,
Preparing a collagen hydrogel solution by adding riboflavin phosphate (RFP) to a mixture containing collagen and a neutralizing solution; Forming a preliminary collagen hydrogel by irradiating the collagen hydrogel solution with blue light having a wavelength range of 450 to 460 nm for 1 to 60 minutes; and incubating the pre-collagen hydrogel at a temperature of 35 to 40° C. for 2 to 4 hours to form a collagen hydrogel.
The step of preparing the collagen hydrogel solution is,
Preparing a neutralizing solution of pH 4 to 9;
Preparing riboflavin phosphate (RFP) with a concentration of 5 to 15 mg/ml by diluting in PBS;
Preparing a mixture by mixing the neutralization solution and collagen at a volume ratio of 4:1 to 1:4; and
Collagen hydrogel comprising; mixing 0.01 to 0.1% by weight of riboflavin phosphate (RFP) with the mixture to prepare a collagen hydrogel solution.
delete A cosmetic set product that promotes the creation of cross-links in the collagen matrix,
A first cosmetic composition corresponding to lotion, skin, wrinkle improvement cream, eye cream, or face oil, and a second cosmetic composition mixed with 0.01 to 0.1% by weight of riboflavin phosphate (RFP) based on the total weight of the first cosmetic composition. Cosmetics comprising the composition; and
It includes a light irradiation device capable of irradiating blue light having a wavelength range of 450 to 460 nm to the object to which the cosmetic is applied,
A cosmetic set product that promotes the creation of cross-links in the collagen matrix, which can improve the degree of cross-linking by irradiating blue light when cross-linking the collagen matrix.

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