WO2021086081A1

WO2021086081A1 - Manufacture of photo-crosslinkable biodegradable tissue adhesive

Info

Publication number: WO2021086081A1
Application number: PCT/KR2020/014955
Authority: WO
Inventors: 양승윤; 박삼대; 이혜선
Original assignee: 부산대학교 산학협력단; 주식회사 에스엔비아
Priority date: 2019-10-31
Filing date: 2020-10-31
Publication date: 2021-05-06

Abstract

The present invention relates to the manufacture of a photo-crosslinkable biodegradable tissue adhesive, and provides a novel hyaluronic acid compound having excellent elongation and excellent adhesive force even in skin and mucosal tissues, in which the physical properties of the adhesive can be adjusted by mixing HAMA with a photo-crosslinkable HA having a different photo-crosslinkable length, and thus, the adhesive can be used as a medical adhesive that can be used in various sites of a body.

Description

Manufacture of photo-crosslinked biodegradable tissue adhesive

The present invention relates to a photo-crosslinkable biodegradable tissue adhesive and a method of manufacturing the same.

Medical tissue adhesives are used for the purpose of healing wounds by protecting the hemostasis, bonding, or treatment area of the wound.

Conventional cyanoacrylate-based tissue adhesives show fast adhesion performance, but their adhesion and flexibility are poor on wet surfaces, and toxic by-products such as formaldehyde are generated during the decomposition process, resulting in low biocompatibility. In addition, since the formed adhesive layer is opaque and uneven, there has been a limitation that it is difficult to apply to a surface requiring high light transmittance, such as an eyeball surface.

In addition, fibrin-based tissue adhesives using the blood coagulation process in the body have high biocompatibility, but have a disadvantage of low adhesion to mucous membranes and wet tissues.

Recently, a photo-crosslinked tissue adhesive capable of maintaining high adhesion to the tissue by forming a hydrogel on the fly by light such as UV is attracting attention. In general, it is possible to achieve high adhesion to a desired tissue by forming a hydrogel by introducing a photo-crosslinking group into a water-soluble polymer to prepare a photo-crosslinking polymer, and irradiating light to a solution obtained by dissolving the photo-crosslinking group.

In particular, hyaluronic acid (HA), which is a constituent of various tissues in the human body, can help in wound recovery, and thus can help not only wound closure but also tissue regeneration with the introduction of the optical crosslinking group.

However, hyaluronic acid methacrylate (HAMA), which is a conventional photo-crosslinked HA, has a problem that it can be used only in limited fields due to poor flexibility and difficulty in controlling physical properties.

Therefore, there is a need for research on a light-crosslinked tissue adhesive that is flexible and can easily control the physical properties of the adhesive.

An object of the present invention is to provide a hyaluronic acid compound having excellent elongation and excellent adhesion to skin and mucous membranes.

In addition, another object of the present invention is to provide a hydrogel or biodegradable tissue adhesive formed through a photocrosslinking reaction using the hyaluronic acid compound.

In addition, another object of the present invention is to provide a biodegradable tissue adhesive in which the hyaluronic acid compound and the hyaluronic acid compound having a different photocrosslink length are introduced in order to control the physical properties of the biodegradable tissue adhesive.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1).

[Formula 1]

In Formula 1,

n is an integer of 1 to 10,000, and X ₁ to X ₄ may be the same or different, respectively, and are OH or a substituent of Formula 1-1.

[Formula 1-1]

In Formula 1-1,

p or q are each an integer of 0 to 10.

In addition, the present invention provides a hydrogel comprising the compound of Formula 1 and formed through a photocrosslinking reaction.

In addition, the present invention provides a biodegradable tissue adhesive comprising the compound of Formula 1 above.

In addition, the present invention provides a composition comprising further mixing a hyaluronic acid methacrylate (HAMA) compound with the compound of Formula 1 above.

In addition, the present invention provides a hydrogel comprising the composition, characterized in that formed through a photo-crosslinking reaction.

In addition, the present invention provides a biodegradable tissue adhesive comprising the composition.

Hyaluronic acid butylacrylate (HABA) prepared according to the present invention has excellent elongation and excellent adhesion to skin and mucous membrane tissues.

In addition, it is possible to adjust the physical properties of the adhesive by mixing hyaluronic acid methacrylate (HAMA), which is a light-crosslinking type HA, and HA having a different light-crosslinking length, so that it can be used as a medical adhesive that can be used on various parts of the body. It can be used as a medical hemostatic agent, wound coating agent, anti-adhesion agent, cell culture support, 3D printer bioink, and bio-coating material.

1 is an NMR image of HAMA.

2 is an NMR image of HAPA.

3 is a diagram showing the elongation of HAMA, HABA, and HAPA.

4 is a view showing the change in tensile strength according to the mixing ratio of HAMA and HABA.

5 is a view showing the change in tensile strength according to the mixing ratio of HAMA and HAPA.

6 is a diagram showing the results of a skin adhesion test of an adhesive in which HAMA, HAMA and HAPA are mixed.

FIG. 7 is a diagram showing the results of a test result of adhesion of an adhesive mixed with HAMA and HAPA to the ocular mucosa.

Hereinafter, the present invention will be described in detail.

The present inventors synthesized a material of hyaluronic acid butylacrylate (HABA), which is excellent in elongation and tissue adhesion, and is a light cross-linking type HA such as hyaluronic acid methacrylate (HAMA) and the HABA. It is possible to prepare tissue adhesives with desired adhesive properties and physical properties by mixing HAs with different crosslinking lengths, so that medical adhesives that can be used on various parts of the body, medical hemostatic agents, wound coating agents, anti-adhesion agents, cell culture supports, 3D printers The present invention was completed by finding that it can be usefully used as a bio-ink and a bio-coating material.

The present invention provides a compound represented by the following formula (1).

[Formula 1]

In Formula 1,

[Formula 1-1]

In Formula 1-1,

p or q are each an integer of 0 to 10.

At this time, X ₁ to X ₄ may include at least one or more substituents of Formula 1-1, the rest may be hydroxy groups, and the substitution rate of the substituents of Formula 1-1 may be 1 to 400%.

In particular, in Formula 1, n is an integer of 1 to 10,000, X ₁ to X ₄ includes at least one substituent of Formula 1-1, and p or q in Formula 1-1 is an integer of 0 to 10, respectively. It is desirable.

As described above, Chemical Formula 1 is characterized by having an ethylenically unsaturated group in the hyaluronic acid repeating unit excellent in biocompatibility.

In addition, the compound represented by Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

In Formula 2, n is an integer of 1 to 10,000.

In addition, the present invention provides a hydrogel comprising any one of the compounds represented by Chemical Formulas 1 to 2, and is formed through a photocrosslinking reaction.

The hydrogel may be used as a physiologically active material or a carrier for a drug delivery system, or as an implant material for tissue regeneration and filling, but is not limited thereto.

In addition, the present invention provides a biodegradable tissue adhesive comprising any one of the compounds represented by Chemical Formulas 1 to 2.

At this time, the compound is a photo-crosslinkable biodegradable tissue adhesive capable of maintaining a high adhesion to the tissue by forming a hydrogel through a crosslinking reaction by light. At this time, in the photo-crosslinking reaction, as well as the crosslinking reaction by UV, if a photoinitiator having a different absorption wavelength is used, it may be possible to form a hydrogel according to the photo-crosslinking reaction in light of other wavelengths including visible light.

In addition, the biodegradable tissue adhesive is made of medical adhesive, medical hemostatic agent, wound coating agent, anti-adhesion agent, medical hemostatic agent, wound coating agent, anti-adhesion agent, cell culture support, 3D printer bioink, and bio-coating material. It may be used for any one or more selected purposes from the group, but is not limited thereto.

Conventional photo-crosslinked hyaluronic acid methacrylate (HAMA) has been used in limited fields due to lack of flexibility during photocrosslinking. In order to improve this, hyaluronic acid butylacrylate (HABA) or hyaluronic acid pentacrylate (HAPA) with excellent elongation is mixed to adjust the mechanical properties of the hydrogel tissue adhesive to the moving part. The range of use can be broadened, and through this, it is expected to increase the adhesion of the tissue.

Accordingly, the present invention provides a composition characterized in that the hyaluronic acid methacrylate (HAMA) compound is further mixed with the compound represented by the following formula (1).

[Formula 1]

In Formula 1,

[Formula 1-1]

In Formula 1-1,

p or q are each an integer of 0 to 10.

In addition, the compound represented by Formula 1 may be a composition represented by Formula 2 or Formula 3.

[Formula 2]

In Formula 2, n is an integer of 1 to 10,000.

[Formula 3]

In Formula 3, n is an integer of 1 to 10,000.

In this case, the HAMA and the compound represented by Chemical Formula 1 may be mixed in a weight ratio of 10:0 to 5:5, and preferably in a weight ratio of 7:3, but are not limited thereto.

According to an embodiment of the present invention, the mechanical properties can be adjusted according to the mixing ratio of each compound of HAMA and HABA or HAMA and HAPA, and the flexibility can be increased than that of conventional HAMA, so that the range of use can be expanded to a moving part. I can.

In addition, it was confirmed that the mixed composition of HAMA and HAPA can have excellent adhesion to not only skin but also mucosal tissues than simple HAMA.

The methacrylate, butyl acrylate, and pentaacrylate are photo-crosslinking agents and react with the hyaluronic acid to obtain a photo-crosslinkable hydrogel. The "photocrosslinking agent" refers to a compound capable of inducing a radical reaction in a reaction system by forming a radical by light irradiation, and a compound that is generally used as a photocrosslinker or a photoinitiator may be used as a photocrosslinker of the present invention, but , It is more preferable to use a photocrosslinking agent or a photoinitiator that is not toxic or weak in vivo.

In this case, the composition is a photo-crosslinkable biodegradable tissue adhesive capable of maintaining high adhesion to the tissue by forming a hydrogel by crosslinking reaction by light. At this time, in the photo-crosslinking reaction, as well as the crosslinking reaction by UV, if a photoinitiator having a different absorption wavelength is used, it may be possible to form a hydrogel according to the photo-crosslinking reaction in light of other wavelengths including visible light.

In the present invention, various light-crosslinking groups of different lengths were introduced to HA, which is a component of various tissues in the human body, and which promotes tissue regeneration while being transparent, to develop a flexible and transparent tissue adhesive that has excellent adhesion to not only skin but also mucosal tissues. . In particular, HA-Butylaacrylate (HABA) showed superior elongation and adhesion properties than other photo-crosslinked HA derivatives. In addition, by mixing hyaluronic acid methacrylate (HAMA), which is a light-crosslinking type HA, and HA having a different light-crosslinking length, to adjust the length of the light-crosslinking group in the HA, A tissue adhesive having desired adhesive properties and physical properties could be prepared.

Since the physical properties of the adhesive can be freely adjusted as described above, it is possible to develop a medical adhesive that can be used on various parts of the body. In addition, the above technology can be applied to medical hemostatic agents, wound coating agents, anti-adhesion agents, cell culture supports, 3D printer bioinks, and biocoating materials, but any application is possible as long as it can be used as a bio-adhesive without limitation. Do.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, to those of ordinary skill in the art to which the present invention pertains. It will be self-evident.

<합성예 1> 히알루론산 부틸아크릴레이트(Hyaluronic acid butylacrylate; HABA)의 합성<Synthesis Example 1> Synthesis of hyaluronic acid butylacrylate (HABA)

[Scheme 1]

As in Reaction Scheme 1, 10 g (26 mmol) of hyaluronic acid is dissolved in 100 ml of purified water and cooled to 0-5°C. 52 mmol of 3-butenoic anhydride and 100 ml of 3M NaOH were added thereto, followed by stirring for 2 days. The reaction product was precipitated in ethanol, purified, and dried in vacuo to prepare hyaluronic acid butylacrylate (HABA). Yield: 85%, ¹ H-NMR (300 MHz, D ₂ O): δ(ppm)= 5.9, 5.3, 5.2 (3H, CH=CH ₂ ), 4.5-4.3 (2H, CH ₂ ), 3.8-3.0 (10H, CH), 2.9 (2H, CH ₂ ), 1.8 (3H, CH ₃ ).

<합성예 2> 히알루론산 메타크릴레이트(Hyaluronic acid methacrylate; HAMA)의 합성<Synthesis Example 2> Synthesis of hyaluronic acid methacrylate (HAMA)

10 g (26 mmol) of hyaluronic acid was dissolved in 100 ml of purified water and cooled to 0-5 °C. After adding 104 mmol of methacrylic anhydride and 100 ml of 3M NaOH, the mixture was stirred for 2 days. The reaction product was precipitated in ethanol, purified, and vacuum-dried to prepare hyaluronic acid methacrylate (HAMA) (see FIG. 1).

<합성예 3> 히알루론산 펜타크릴레이트(Hyaluronic acid Pentacrylate; HAPA)의 합성<Synthesis Example 3> Synthesis of Hyaluronic acid Pentacrylate (HAPA)

10 g (26 mmol) of hyaluronic acid was dissolved in 100 ml of purified water and cooled to 0-5 °C. After adding 156 mmol of 4-pentenoic anhydride and 100 ml of 3M NaOH, the mixture was stirred for 2 days. The reaction product was precipitated in ethanol, purified, and vacuum-dried to prepare hyaluronic acid pentacrylate (HAPA) (see FIG. 2).

<실시예 1> HAMA, HABA, HAPA 의 물성 측정<Example 1> Measurement of physical properties of HAMA, HABA, and HAPA

The elongation of HAMA, HABA, and HAPA synthesized through Synthesis Examples 1 to 3 was measured through a tensile test.

Tensile tests were performed on a test piece molded in a dogbone shape according to the ASTM method at a rate of 1 mm/min using a universal testing machine of AND Corporation. The elongation was analyzed by measuring the strain at the fracture point of the specimen.

As a result, as shown in FIG. 3, the elongation tends to increase as the length of the optical crosslinking increases. It was confirmed that HABA can increase three times or more from 7.5 mm before stretching to 25.5 mm after stretching, and compared with HAMA, HABA and HAPA showed excellent elongation.

<실시예 2> HABA를 이용한 히알루론산 메타크릴레이트(Hyaluronic acid methacrylate; HAMA)의 물성 조절<Example 2> Adjustment of physical properties of hyaluronic acid methacrylate (HAMA) using HABA

The mixing ratios of HAMA and HABA synthesized through Synthesis Examples 1 and 2 were mixed at different weight ratios of 10:0, 7:3 and 5:5, and then the tensile strength was measured.

Tensile tests were conducted using AND's universal testing machine, and the specimen molded in a dogbone shape according to the ASTM method was measured at a speed of 1 mm/min. curve) was measured.

As a result, as shown in FIG. 4, it was confirmed that the mechanical strength was changed according to the mixing ratio of HAMA and HABA.

<실시예 3> 히알루론산 펜타크릴레이트(Hyaluronic acid pentacrylate; HAPA)를 이용한 히알루론산 메타크릴레이트(Hyaluronic acid methacrylate; HAMA)의 물성 조절<Example 3> Adjustment of physical properties of hyaluronic acid methacrylate (HAMA) using hyaluronic acid pentacrylate (HAPA)

After mixing by varying the mixing ratio of HAMA and HAPA synthesized through Synthesis Examples 1 and 3 in a weight ratio of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5, tensile The strength was measured.

Tensile tests were performed on a specimen molded in a dogbone shape according to the ASTM method using a universal testing machine from AND at a speed of 1 mm/min. curve) was measured.

As a result, as shown in FIG. 5, it was confirmed that the mechanical strength was changed according to the mixing ratio of HAMA and HAPA.

<실시예 4> 동적환경에서 HAMA와 HAPA가 혼합된 접착제의 접착력 테스트<Example 4> Adhesion test of an adhesive mixed with HAMA and HAPA in a dynamic environment

In order to evaluate the adhesion performance of the adhesive mixed with HAMA and HAPA, a skin adhesion test of the adhesive mixed with HAMA, HAMA and HAPA at 7:3 was conducted.

On the surface of pig skin, HAMA or HAMA and HAPA are mixed at 7:3, and the photoinitiator, lithium phenyl-2,4,6-triethylbenzoyl phosphinate (Lithium phenyl-2,4,6-trimethylbenzoyl phosphinate; LAP) After applying the adhesive solution to which 0.1 wt% was added, UV was irradiated for 5 seconds, and the adhesive strength was confirmed in a dynamic environment by bending.

As a result, as shown in FIG. 6, when the HAMA single adhesive was folded over 90° (bending test), it was detached due to low flexibility, but the HAMA and HAPA mixed adhesive adhered to a bending of about 90° due to an increase in flexibility. It was confirmed that it was maintained.

<실시예 5> 점막조직에서 HAMA와 HAPA가 혼합된 접착제의 접착력 및 조직 회복력 테스트<Example 5> Adhesion and tissue resilience test of an adhesive mixed with HAMA and HAPA in mucosal tissue

In order to check the tissue adhesion retention and resilience in a humid environment, an adhesive strength test of the adhesive mixed with HAMA and HAPA at 7:3 was performed.

Using a surgical knife to artificially incise the rabbit's eye mucosa, and mix HAMA and HAPA at 7:3, and lithium phenyl-2,4,6-triethylbenzoyl phosphinate (Lithium phenyl-2,4) as a photoinitiator. ,6-trimethylbenzoyl phosphinate; LAP) 0.1 wt% added adhesive solution was applied, UV irradiated for 5 seconds, and visually confirmed adhesion.

As a result, it was confirmed that, as shown in FIG. 7, when an adhesive in which HAMA and HAPA were mixed at 7:3 was applied, adhesion to the mucous membrane could be maintained during wound recovery, and thus the incision was stably repaired. On the other hand, it was confirmed that the uneven tissue was recovered by exposing the incision in the group to which the adhesive was not applied.

Claims

Compound represented by the following formula (1):

[Formula 1]

In Formula 1,

n is an integer of 1 to 10,000, and X 1 to X 4 may be the same or different, respectively, and are OH or a substituent of Formula 1-1.

[Formula 1-1]

In Formula 1-1,

p or q are each an integer of 0 to 10.
The method of claim 1,

In Formula 1, n is an integer of 1 to 10,000, X 1 to X 4 include at least one substituent of Formula 1-1, and p or q in Formula 1-1 is an integer of 0 to 10, respectively. Compound.
The method of claim 1,

The compound represented by Formula 1,

A compound characterized by being a compound represented by the following formula (2):

[Formula 2]

In Formula 2, n is an integer of 1 to 10,000.
A hydrogel comprising the compound of any one of claims 1 to 3 and formed through a photocrosslinking reaction.
A biodegradable tissue adhesive comprising the compound of any one of claims 1 to 3.
The method of claim 5,

The compound is a biodegradable tissue adhesive, characterized in that the crosslinking reaction by light to form a hydrogel (hydrogel).
A composition, characterized in that a hyaluronic acid methacrylate (HAMA) compound is further mixed with the compound represented by the following formula (1):

[Formula 1]

In Formula 1,

n is an integer of 1 to 10,000, and X 1 to X 4 may be the same or different, respectively, and are OH or a substituent of Formula 1-1.

[Formula 1-1]

In Formula 1-1,

p or q are each an integer of 0 to 10.
The method of claim 7,

The compound represented by Formula 1,

A composition characterized in that it is a compound represented by the following formula (2) or formula (3):

[Formula 2]

In Formula 2, n is an integer of 1 to 10,000.

[Formula 3]

In Formula 3, n is an integer of 1 to 10,000.
The method of claim 7,

The composition characterized in that the HAMA and the compound represented by Formula 1 are mixed in a weight ratio of 10:0 to 5:5.
A hydrogel comprising the composition of any one of claims 7 to 9 and formed through a photocrosslinking reaction.
Biodegradable tissue adhesive comprising the composition of any one of claims 7 to 9.
The method of claim 11,

The composition is a biodegradable tissue adhesive, characterized in that a crosslinking reaction occurs by light to form a hydrogel.