KR20200101818A - Dual-crosslinked (temperature sensitive, light sensitive) biocompatible chitosan hydrogel composition and its manufacturing method - Google Patents

Abstract

The present invention relates to a hydrogel composition and a method for manufacturing a hydrogel capable of double crosslinking of temperature crosslinking and photocrosslinking using the hydrogel composition.

Description

Dual-crosslinked (temperature sensitive, light sensitive) biocompatible chitosan hydrogel composition and its manufacturing method}

The present invention relates to a hydrogel composition capable of having a double-crosslinked structure of temperature (heat) crosslinking, photocrosslinking, or temperature crosslinking and photocrosslinking, and a method of preparing the hydrogel composition.

Since bioprinting requires distribution of a cell-containing medium, it is important that the material is cytocompatible in bioprinting. To this end, hydrogels using materials such as gelatin, gelatin/chitosan, gelatin/alginate, gelatin/fipronectin, Lutrol F127/alginate, and alginate are used for bioprinting. The hydrogel or a mixture of hydrogels and cells used for bioprinting is also referred to as'bioink'.

However, in bioprinting, it is important to prevent contamination from foreign substances or bacteria when culturing or printing cells, and further, satisfies the sophistication and detail of the biostructure so that it can be applied to the human body, and also prevents cytotoxicity or cellular compatibility. It is important to mold structures that are sufficiently satisfactory.

Hydrogels have excellent hydrophilicity, so they can easily absorb water and change their strength and shape, so they are used as a tissue engineering scaffold or drug delivery. The hydrogel absorbs a large amount of water in an aqueous solution or in an aqueous environment due to the hydrophilicity of its constituent materials, and it swells but does not dissolve due to a crosslinked structure. Therefore, hydrogels having various shapes and properties can be made depending on the composition and the manufacturing method, and they generally contain a large amount of moisture, and are characterized by having intermediate properties between liquid and solid.

On the other hand, gelatin, which is widely used as a biocompatible polymer for hydrogels, is a protein obtained by partial hydrolysis of collagen, a major protein component of connective tissues such as animal bones, cartilage, and leather, and has high biocompatibility and non-toxic biodegradability. Have. Gelatin gives viscosity even at a relatively low temperature and concentration, and gelatin solution forms a clear and elastic thermoreversible gel when cooled, but it dissolves easily in an aqueous solution, so formaldehyde or glutaaldehyde is used to increase its stability. Crosslinking with chemicals such as rutaaldehyde is being used. However, if these crosslinking agent components remain inside even a small amount, they may exhibit cytotoxicity due to this and may exhibit toxicity to surrounding organs caused by them after implantation in the body.

In this regard, Korean Patent Application Publication No. 2019-0016535 discloses a hydrogel composition having high viscosity properties without a chemical crosslinking agent.

Korean Patent Registration No. 10-1957415 Republic of Korea Patent Publication No. 2019-0016535

The present invention is to provide a hydrogel composition capable of having a temperature (thermal) crosslinking, a photocrosslinking, or a double crosslinked structure of temperature crosslinking and photocrosslinking, and a method of preparing the hydrogel composition.

In order to solve the above problem,

The present invention in one embodiment,

It provides a hydrogel composition comprising a chitosan compound having a repeating unit represented by the following formulas 1 to 3:

[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,

R ₁ is hydrogen or an alkyl group having 1 to 4 carbon atoms,

이며,R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

Is,

R ₅ is hydrogen or an alkyl group having 1 to 4 carbon atoms,

p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.20≤q≤0.50, 0.20≤r≤0.50, p>0 Satisfies the phosphorus condition.

In addition, the present invention is a method for producing a hydrogel composition comprising the step of preparing a chitosan compound having a repeating unit represented by the following Formulas 1 to 3 from a mixture containing a chitosan solution, an epoxy compound, and a methacrylic anhydride in an embodiment. Provides:

[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,

R ₁ is hydrogen or an alkyl group having 1 to 4 carbon atoms,

이며,R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

Is,

R ₅ is hydrogen or an alkyl group having 1 to 4 carbon atoms,

p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.20≤q≤0.50, 0.20≤r≤0.50, p>0 Satisfies the phosphorus condition.

By using the hydrogel composition prepared by substituting the hydroxy group and amine group of chitosan according to the present invention, it is possible to prepare a chitosan hydrogel that is sensitive to body temperature and a wide range of light. Specifically, the hydrogel according to the present invention can be crosslinked without an additional crosslinking agent at a temperature similar to body temperature by adding a soluble group to chitosan at neutral pH by substituting a hydroxy group of chitosan to impart temperature reactivity. In addition, the hydrogel according to the present invention can improve the strength of the hydrogel and impart cell adhesion by substituting the amine group of chitosan with an acrylic anhydride, and can be crosslinked under a wide light range (or visible light). Accordingly, side effects due to ultraviolet rays can be minimized, and a light and useful hydrogel can be provided.

In addition, since the hydrogel according to the present invention has viscosity that can be used with a syringe and tube, it can be applied to various delivery methods, so it can have printability, so it can be used as an ink for 4D printing. In addition, the shape can be maintained with an appropriate viscosity and adhesion even in water at room temperature, and light crosslinking is possible even after temperature crosslinking.

1 is a schematic diagram of the utilization of HBC-MA for 4D bioprinting using the hydrogel of the present invention: (A) A 3% HBC-MA solution capable of photo-crosslinking reaction is set to pH 7.4. Put the solution in the extruded cylinder and print it directly underwater. The HBC-MA solution is not diluted with water, retains its extruded shape and exhibits a red light.
(B) The HBC-MA exhibits temperature reactivity in water maintained at 37 to 40°C, and the temperature crosslinked HBC-MA hydrogel has a pink color. At this time, if the internal pores of the freeze-dried hydrogel are observed using SEM, the pore size is large so that the cells can move freely.
(C) It shows a portion exposed to visible light, and a photocrosslinking phenomenon due to visible light occurs even in a state in which a reaction is observed by temperature crosslinking. The visible light crosslinked hydrogel has a yellow color. At this time, when the internal pores of the freeze-dried hydrogel are observed using SEM, the pore size is small and the cells cannot pass through, and the cells are captured by cell adhesion.
2 is a graph showing 1H NMR of chitosan, HBC and HBC-MA according to an embodiment of the present invention.
3 shows FTIR spectra of chitosan, HBC and HBC-MA according to an embodiment of the present invention.
4A and 4B show the rheological analysis of 3% by weight HBC-MA and the gel point at which G'and G" intersect in an example of the present invention.
5A and 5B show the mechanical properties of HBC-MA having various pH and visible light exposure time in an embodiment of the present invention (*p<0.05, ***p<0.001).
6 shows the results of measuring the viability of NIH3T3 cells by WST-1 analysis in the examples of the present invention. Error bars represent the SE of repeated measurements for each sample (n=6 for each group).
7A to 7C are SEM images inside a temperature crosslinked HBC-MA hydrogel (FIG. 7A ), a SEM image inside a double crosslinked HBC-MA hydrogel (FIG. 7B ), and It is an SEM image (Fig. 7c) magnified 700 times inside the crosslinked HBC-MA hydrogel.
8A to 8E are photographs showing a process of crosslinking by printing HBC-MA in order to test the possibility of 4D printing in an embodiment of the present invention.

In the present invention, various modifications may be made and various embodiments may be provided, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

In the present invention, terms such as "comprises" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or a combination thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance.

In the present invention, "4D (4th demension) printing" refers to a technology for 3D printing an object that changes or manufactures a shape by itself when a predetermined time or arbitrary environmental condition is satisfied, and specifically It means programming materials that can self-deform, such as physical and biological materials, to change their shape and properties. Here, 4D has a different meaning from the four-dimensional space of mathematics or physics.

In the present invention, "alkyl group" may mean a functional group derived from a linear or branched saturated hydrocarbon. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, sec -Butyl group (sec-butyl group), t-butyl group (tertbutyl group), n-pentyl group (n-pentyl group), 1,1-dimethylpropyl group (1,1-dimethylpropyl group), 1,2- Dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, n-hexyl group, 1-methyl-2-ethylpropyl group, 1-ethyl-2-methylpropyl group, 1 ,1,2-trimethylpropyl group, 1-propylpropyl group, 1-methylbutyl group, 2-methylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group , 1,3-dimethylbutyl group, 2,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, and the like.

Hereinafter, the present invention will be described in detail.

Chitosan, a biocompatible polymer that is widely used among materials used for living organisms such as 4D printing and bioprinting, has non-toxicity, biodegradability, and biocompatibility, but has a disadvantage of aggregation due to its insoluble property at neutral pH. Thus, in order to use chitosan in neutral to expand its use to various fields (medical, etc.), in the present invention, crosslinking can occur at a temperature similar to body temperature, and light (visible light, etc.) in a wavelength range that is harmless compared to UV is used as a medium. Thus, crosslinking occurs to provide a hydrogel capable of double crosslinking.

Specifically, the present invention provides a hydrogel composition comprising a chitosan compound having a repeating unit represented by the following Formulas 1 to 3:

[Formula 1]

[Formula 2]

[Formula 3]

이며, R₅는 수소, 탄소수 1 내지 4의 알킬기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.20≤q≤0.50이며, 0.20≤r≤0.50이되, p>0인 조건을 만족한다.In Formulas 1 to 3, R ₁ is hydrogen, an alkyl group having 1 to 4 carbon atoms, and R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

And R ₅ is hydrogen, an alkyl group having 1 to 4 carbon atoms, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, and 0.20≤ The condition that q≤0.50, 0.20≤r≤0.50, and p>0 is satisfied.

이며, R₅는 수소 또는 메틸기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.20≤q≤0.30이며, 0.10≤r≤0.30이되, p>0인 조건을 만족할 수 있다.As an example, R ₁ is hydrogen, a methyl group or an ethyl group, and R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

And R ₅ is hydrogen or a methyl group, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, and 0.20≦q≦0.30, 0.10≤r≤0.30, but a condition of p>0 may be satisfied.

이며, R₅는 수소 또는 메틸기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.30≤q≤0.40이며, 0.30≤r≤0.50이되, p>0인 조건을 만족할 수 있다.In addition, as an example, the R ₁ is hydrogen, a methyl group or an ethyl group, and R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

And R ₅ is hydrogen or a methyl group, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.30≦q≦0.40, 0.30≤r≤0.50, but a condition of p>0 may be satisfied.

For example, Formula 1 may include chitosan.

For example, the chitosan compound may include a repeating unit represented by Formula 4:

[Formula 4]

이고, R₇은 R₅는 수소 또는 메틸기이다.In Formula 4, R ₆ are each independently hydrogen or

And R ₇ is R ₅ is hydrogen or a methyl group.

The chitosan compound may be characterized by having a structure in which the repeating units represented by Formulas 1 to 3 are crosslinked by either light or heat.

The hydrogel composition according to the present invention may be a chitosan hydrogel composition sensitive to temperature and light similar to body temperature by having a crosslinked structure by substituting hydroxy groups and amine groups of chitosan.

The hydrogel composition according to the present invention can be crosslinked without an additional crosslinking agent at a temperature similar to body temperature by adding a soluble group to chitosan at neutral pH by substituting a hydroxy group of chitosan to impart temperature reactivity. In addition, by substituting the amine group of chitosan with an acrylic anhydride, the strength of the hydrogel can be improved, cell adhesion can be imparted, and crosslinking can be performed under visible light. Accordingly, side effects due to ultraviolet rays can be minimized, and a light and useful hydrogel can be provided.

The chitosan compound may maintain a crosslinked structure at a temperature of 30 to 50°C. For example, the chitosan compound may maintain a crosslinked structure at a temperature of 30 to 45°C, 30 to 40°C, 30 to 35°C, 35 to 50°C, 40 to 50°C, or 45 to 50°C.

The chitosan compound may maintain a crosslinked structure under light irradiation conditions having a wavelength of 300 nm to 800 nm. For example, the chitosan compound is in the range of 300 to 700 nm, 300 to 600 nm, 300 to 500 nm, 300 to 400 nm, 400 to 800 nm, 500 to 800 nm, 600 to 800 nm, or 700 to 800 nm. The crosslinked structure can be maintained under light irradiation conditions having a wavelength.

The chitosan may have a molecular weight of 50,000 to 190,000 Da. For example, the molecular weight of the chitosan may be 50,000 to 150,000 Da, 50,000 to 120,000 Da, 50,000 to 100,000 Da, 50,000 to 80,000 Da, 80,000 to 190,000 Da, 120,000 to 190,000 Da, or 150,000 to 190,000 Da.

The hydrogel according to the present invention has good viscosity and can be applied to various delivery methods by having a viscosity that can be used with a syringe and a tube, so it can have printability, so it can be used as an ink for 4D printing. In addition, the shape can be maintained with an appropriate viscosity and adhesion even in water at room temperature, and light crosslinking is possible even after temperature crosslinking.

For example, FIG. 1 is a schematic diagram showing the printing process when the hydrogel composition of the present invention is used for 4D printing: (A) The hydrogel composition is put in an extruded cylinder and printed directly in water, and the hydrogel composition is Undiluted, retains its extruded shape and exhibits red light.

(B) The hydrogel composition exhibits temperature reactivity in water maintained at 30 to 50°C, and the temperature crosslinked hydrogel has a pink color. At this time, the size of the internal pores (pores) of the hydrogel is large enough to allow the cells to move freely when the cells are attached.

(C) It shows a portion of the hydrogel exposed to visible light, and a photo-crosslinking phenomenon due to visible light may occur even when a reaction is observed by temperature crosslinking. The visible light crosslinked hydrogel exhibits yellow color, and since the hydrogel has a small size of the inner pores that the cells cannot pass through, the cells are captured when the cells adhere.

In addition, the present invention provides a method for preparing a hydrogel composition comprising the step of preparing a chitosan compound having a repeating unit represented by the following Formulas 1 to 3 from a mixture containing a chitosan solution, an epoxy compound, and a methacrylic anhydride:

[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,

이며, R₅는 수소, 탄소수 1 내지 4의 알킬기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.20≤q≤0.50이며, 0.20≤r≤0.50이되, p>0인 조건을 만족한다.R ₁ is hydrogen, an alkyl group having 1 to 4 carbon atoms, and R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

And R ₅ is hydrogen, an alkyl group having 1 to 4 carbon atoms, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, and 0.20≤ The condition that q≤0.50, 0.20≤r≤0.50, and p>0 is satisfied.

이며, R₅는 수소 또는 메틸기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.20≤q≤0.30이며, 0.10≤r≤0.30이되, p>0인 조건을 만족할 수 있다.As an example, R ₁ is hydrogen, a methyl group or an ethyl group, and R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

And R ₅ is hydrogen or a methyl group, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, and 0.20≦q≦0.30, 0.10≤r≤0.30, but a condition of p>0 may be satisfied.

이며, R₅는 수소 또는 메틸기이고, p, q 및 r은 키토산 화합물에 포함된 화학식 1 내지 3으로 나타내는 반복단위의 몰 분율로서, p+q+r=1이고, 0.30≤q≤0.40이며, 0.30≤r≤0.50이되, p>0인 조건을 만족할 수 있다.In addition, as an example, the R ₁ is hydrogen, a methyl group or an ethyl group, and R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

And R ₅ is hydrogen or a methyl group, p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.30≦q≦0.40, 0.30≤r≤0.50, but a condition of p>0 may be satisfied.

The hydrogel composition is reacted by mixing a chitosan solution and an epoxy compound to replace (epoxylate) a hydroxyl group (OH group) of chitosan (first reaction). The above reaction can impart water solubility and temperature reaction properties to chitosan. Specifically, the first reaction may generate water-soluble chitosan by adding a soluble group to insoluble chitosan at a neutral pH and impart temperature reactivity. At this time, it is maintained in a solution state below room temperature (24° C.), and a hydrogel can be formed at 30 to 50° C. similar to body temperature, and when the temperature is lower than room temperature again, it may return to the solution state.

The epoxy compound may be a compound represented by Formula 6:

[Formula 6]

In Formula 6, R ₉ is hydrogen or an alkyl group having 1 to 4 carbon atoms.

The first reacted chitosan solution is mixed with methacrylic anhydride and reacted to methacrylate an amine group of chitosan (second reaction) to prepare a hydrogel composition comprising a chitosan compound having a repeating unit represented by Formulas 1 to 3. I can. The methacrylic anhydride was performed to compensate for the first reacted chitosan, since it has relatively weak crosslinking strength and low cell adhesion. Accordingly, in order to minimize side effects due to ultraviolet rays, a hydrogel capable of providing a more useful hydrogel can be prepared.

The methacrylic anhydride may be a compound represented by the following formula (5):

[Formula 5]

In Formula 5, R ₈ is hydrogen or an alkyl group having 1 to 4 carbon atoms.

Specifically, the step of preparing a chitosan compound may include reacting a chitosan solution with an epoxy compound to form an intermediate compound including a repeating unit represented by Formulas 1 and 2; And reacting the intermediate compound with methacrylic anhydride to form a compound including a repeating unit represented by Formulas 1 to 3.

Before the step of forming the intermediate compound, a step of adjusting the pH of the chitosan solution to neutral, specifically, to 6 to 8, using a basic compound may be further included. For example, the basic compound is ammonia (NH ₃ ), ammonium hydroxide (NH ₄ OH), magnesium hydroxide (Mg(OH)), potassium hydroxide (KOH), calcium hydroxide (Ca(OH) ₂ ), sodium hydroxide (NaOH). ), barium hydroxide (Ba(OH) ₂ ), aluminum hydroxide (Al(OH) ₃ ), iron hydroxide (Fe(OH) ₂ ), sodium hydrogen carbonate (NaHCO ₃ ), sodium carbonate (NaCO), calcium carbonate (CaCO ₃ ) , Potassium carbonate (K ₂ CO ₃ ), methylamine (CH ₃ NH ₂ ), and aniline (C ₆ H ₅ NH ₂ ) may include at least one selected from the group consisting of.

After preparing the hydrogel composition, the hydrogel composition may be temperature crosslinked. Temperature crosslinking can be carried out at a temperature similar to body temperature and is easily possible without additional crosslinking agents. The temperature crosslinking may be performed at a temperature range of 30 to 50°C. For example, the temperature crosslinking is performed at 30 to 45°C, 30 to 40°C, 30 to 35°C, 35 to 50°C, 35 to 45°C, 37 to 50°C, 40 to 50°C, or 45 to 50°C It can be.

After the temperature crosslinking, the temperature range may be maintained, and then the temperature of 30 to 50°C may be maintained even during photocrosslinking.

The hydrogel composition is in a liquid state, and when the hydrogel is prepared by temperature crosslinking, it is in a gel state, so its utility is increased compared to when it is in a liquid state, but it is still low in strength, therefore, photocrosslinking is added to prepare a hydrogel. By doing this, it was intended to increase the strength and diversify its use.

Accordingly, after the temperature crosslinking, photocrosslinking may be performed. Photocrosslinking the hydrogel composition may be performed by mixing a photoinitiator with the hydrogel composition and irradiating visible light.

During the photocrosslinking, the wavelength of light may range from 300 to 800 nm. For example, the wavelength of the light is in the range of 300 to 700 nm, 300 to 600 nm, 300 to 500 nm, 300 to 400 nm, 400 to 800 nm, 500 to 800 nm, 600 to 800 nm, or 700 to 800 nm. I can. For example, the wavelength of the light may be determined in a required range according to the photoinitiator material, and various ranges of photoinitiators may be used during photocrosslinking according to the present invention.

The photoinitiator is triethanolamine, N-vinyl caprolactam, lithium aryl phosphinate, 2,2-dimethoxy-2-phenylacetophenone, benzyldimethylketal, ethylbenzoin ether, isopropylbenzoin ether, 2,2- Diethoxyacetophenone, 2-hydroxy-2-methyl-1-phenylpropan-1-one, 1-hydroxycyclohexylphenylketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2 -Methylpropiophenone, benzyl benzoate, eosin wire, riboflavin, rose bengal, and benzoyl isobutyl ether may include one or more selected from the group consisting of.

The temperature crosslinked and photocrosslinked hydrogel may have a visual effect of changing color when exposed to visible light, and temperature crosslinking occurs when the pH is neutral and has an ultra-high crosslinking rate for forming a hydrogel at neutral pH. I can.

The hydrogel according to the present invention is suitable for body temperature and pH conditions and can react in a wide wavelength range. As a result, temperature crosslinking occurs within a body temperature range, and a photocrosslinked hydrogel may be formed in the range of 300 to 800 nm. Accordingly, crosslinking occurs within tens of seconds due to an increase in temperature, and a crosslinked hydrogel can be formed at a temperature having reversibility so that crosslinking is released when the temperature is low, and at this time, the pore space inside the hydrogel is a wide and low adhesive. I can. In addition, a photo-crosslinked hydrogel may be formed by light in the range of 300 to 800 nm, specifically, visible light, in which case the pore space inside the hydrogel is narrowed and may be structurally very elastic, and has high elasticity and adhesion. Can represent.

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[ Example ]

1. Substance

Chitosan (MW 50-190 kDa, DD 75-85%), methacrylic anhydride, 1,2-epoxybutane, EosinY (99% dye content), TEA (triethanol amine), NVP (1-vinyl-2-p) Lolidinone, sodium hydroxide inhibitor, >99%) was purchased from Sigma Co.Ltd. All other chemicals used in this example are of analytical grade.

2. Hydroxybutyl Methylacrylamide Chitosan ( Hydroxybutyl methylacrylamide chitosan, HBC - MA ) Of manufacture

In order to prepare HBC-MA in this example, first 1 g of chitosan (low molecular weight, Sigma-aldrich) was completely dissolved in 60 mL of 0.1 M HCl. The chitosan solution was adjusted to pH 6 using 5 M KOH. After adjusting the pH, 20 mL of 1,2-epoxybutane was added, and the mixture was stirred well for 24 hours to avoid volatilization of 1,2-epoxybutane. At this time, the hydroxy group of chitosan and the epoxy butane undergo a base catalyst epoxide reaction. The reaction was carried out at 55° C. for 24 hours. After all reactions were completed, 1,2-epoxybutane was removed by centrifugation to prepare a hydroxybutyl chitosan (HBC) solution. The HBC solution was frozen at -20[deg.] C. and then frozen in a freezer for 1 day, and then freeze-dried for about 4 days to prepare pure HBC.

1 g of HBC thus obtained was dissolved in 60 mL of 0.1 M HCl overnight, and then 15 mL of methacrylic anhydride was added. At this time, it was stirred using a magnetic bar, and a rotation speed of 250 rpm was used so that no air bubbles were generated. At this time, the amino group of chitosan and methacrylic anhydride undergo a Michael reaction. After reacting for 4 hours, the pH of the solution was adjusted to 7 using 5 M KOH. It was diluted using a dialysis tube (12 to 14 kDa), and the remaining methacrylic anhydride was removed for a week by replacing distilled water twice a day. After removal of methacrylic anhydride, it was frozen at -20°C, and it was further frozen in a freezer for 1 day, and then freeze-dried for 4 days to obtain pure hydroxybutyl methylacrylamide chitosan (HBC- MA) was obtained.

[ Experimental example ]

1. 1H nuclear magnetic resonance (1H Proton Nuclear Magnetic Resonance , 1H-NMR) spectroscopic measurement

Chemical structures of chitosan (CS), HBC and HBC-MA were analyzed using FTIR-4100 (Jasco, JAPAN), and all samples were 0.25% deuterium in 1% (w/v) of deuterium oxide (D2O). It was dissolved in chloride (Deuterium chloride, DC1) and analyzed using a 500 MHz FT-NMR spectrometer (Varian Ltd., USA).

HBC-MA was dissolved in 0.25% DC1 in 1% (w/v) heavy water (Deuterium Oxide, D2O) by vortexing. As a control, hydroxybutyl-chitosan and chitosan were also prepared at the same concentration in the same solvent. The 1H-NMR spectrum was recorded using a 500 MHz FT-spectrometer, and the results are shown in FIG. 2. The peak of 2.06 ppm is attributed to the three protons of N-acetyl glucosamine, and the peak of 2.99 ppm is attributed to the proton of the glucosamine residue (H-2). Peaks from 3.5 ppm to 4.0 ppm correspond to non-anomeric protons (H-3, H-4, H-5, H-6). In the 1 H NMR spectrum of HBC, a new signal at 0.92 ppm appears due to the substitution of the methyl group of hydroxybutyl. 5.7 and 6.11 ppm are methylene peaks that occur as covalent bonds in methacrylolyl groups replacing methacrylamide. 1.8 ppm was labeled with MA bound to HBC-MA having a methyl proton of the methacryloyl group. This demonstrated that the substitution of chitosan with HBC for hydroxyl-butyl substitution results in the successful addition of methacrylamide to the proton of -NH ₂ . The degree of methacrylate is the integral area of the H3-H6 peak of the C-chitosan residue versus the integral area of 3.3 and 3.8 ppm (H-5) versus the integration of the CH ₂ peak of the methacrylate group at 5.6 and 6.4 ppm. It is the area (H-2). The degree of methacrylation of chitosan after modification using the method by 1 H NMR was 24.8%.

2. FTIR Spectroscopy

The synthesis of HBC-MA could be confirmed through FTIR comparison. FTIR analysis was performed using FTIR-4100 (Jasco, JAPAN) for the chemical structures of chitosan (CS), HBC, and HBC-MA, and the results are shown in FIG. 3.

The broadband in the region of 3247 to 3398 cm ^-1 in chitosan's FTIR corresponds to the superposition of vibrations of OH and NH. The bands of 1024 and 1067 cm ^-1 showed a unique band of 6-OH in chitosan. The 1153 cm ^-1 peak is generally due to the asymmetric COC bridge. The absorption bands of 2911 and 2873 cm ^-1 can be represented by CH symmetric and asymmetric stretching. The band of 1020 to 1060 cm ^-1 mainly representing 6-OH in the FTIR of HBC is less than that of the FTIR chitosan spectrum. New peaks from 2944 to 2892 cm ^-1 and 1585 cm ^-1 indicate the bending and CH elongation of the newly formed CH ₃ groups due to the introduction of 1,2-epoxybutane. It was found that the hydroxybutyl group was mainly substituted with 6-OH. In the FTIR of HBC-MA, a peak elevation of HBC-MA was observed at 1614 cm ^-1 associated with the NH ₂ band. The absorbances of the newly formed amide 3, 2 and 1 bands were consistent at 1306 cm ^-1 , 1542 cm ^-1 and 1710 cm ^-1 . For this reason, it was confirmed that HBC was further substituted with HBC-MA.

3. Rheological ( rheological ) analysis

A rheometer was used to measure the dynamic mechanical properties of the change from solution to hydrogel, and 3% HBC-MA hydrogel was measured using an MCR302 rheometer (Anton Paar Ltd., Austria). Measurement conditions were the storage modulus (G') and loss modulus (G") of the HBC aqueous solution at a heating rate of 1°C/min and a frequency of 1 rad/s, and in this process, the temperature was increased from 4 to 50°C.

The measurement results are shown in FIGS. 4A and 4B, and FIG. 4A shows a rheological analysis of 3% by weight HBC-MA, and FIG. 4B shows a gel point at which G'and G” intersect.

The gel point is defined as the temperature at which the storage modulus (G') and the loss modulus (G") are the same. The loss modulus (G") is higher than the storage modulus (G') at 4°C at the beginning of the graph, but both modulus are very Shows a low number. As the temperature rises, the sol-gel transition occurs and the storage modulus slightly increases. At 27.2°C, G'and G" are the same, 27.2°C is the gel point, and as the temperature rises past the gel point, the storage modulus increases at a faster rate. Conversely, when the temperature decreases, as is known before Studies have confirmed that it can be reversibly changed, which indicates that the gelling conditions of HBC-MA are very similar to the biocompatible environment, ie that the temperature crosslinked-hydrogel can be formed in an environment similar to body temperature. Able to know.

4. Mechanical test

The prepolymer solution was placed in a PDMS mold having a diameter of 8 mm and a height of 2.5 mm, and placed on a slide glass in a Petri dish containing PBS at 40°C. Each sample was exposed to 35 mW/cm ² visible light (450-550 nm) in PBS at 40° C. for 10 seconds, 20 seconds and 30 seconds. In addition, HBC-MA with 15% methacrylation degree was exposed at pH 6.4, 7.4 and 8.4 for 10 seconds, 20 seconds and 30 seconds, respectively.

After taking out the slide glass, the mold was carefully removed and lightly wiped with kimwipe. The test was performed using a texture analyzer (brookfield) with a trigger load of 0.05 N and a test speed of 0.05 mm/s. The compression coefficient was determined as the slope of the linear region corresponding to 5 to 15% strain.

The measurement results are shown in FIGS. 5A and 5B, and FIG. 5A shows the mechanical properties of HBC-MA with different exposure times to visible light, and FIG. 5B shows the mechanical properties of HBC-MA with different pH exposure times. .

The mechanical properties of the hydrogel on the influence of the visible light exposure time and the pH first have an ultra-fast crosslinking rate that forms the hydrogel within 30 seconds. In general, as can be seen from the representative curve of HBC-MA 3% (w/v) at pH 7.4, it can be seen that increasing the visible light exposure time increases the stiffness at all strain rates for all three sample pH concentrations. The compression coefficient was significantly higher at the pH value. This behavior was observed at 10 sec, but was not statistically significant. Likewise, when the same pH value was used at the same time, the coefficient increased sharply in all conditions as the visible light exposure time slightly increased. All conditions were significantly different except for the 10 seconds sample (*p<0.05, ***p<0.001). Error bars represent the standard deviations (SD) of measurements performed on 8 samples.

5. WST -One( water - soluble tetrazolium salt -1) Analysis method

To evaluate the effect of HBC-MA concentration on cell viability, NIH3T3 was incubated with media containing different concentrations of HBC-MA solutions. As a control, a sample cultured in a medium using only fetal bovine serum (FBS) for cytotoxicity of NIH3T3 cells was used.

Specifically, NIH3T3 fibroblasts were cultured and maintained in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were cultured in a 37° C. 5% CO ₂ incubator, and the culture medium was changed daily. To investigate the toxic effect of HBC-MA solution on cell culture, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3 -Benzene disulfonate was used to test the viability of cells (WST-1 analysis). HBC-MA solution (2 mg/mL) was prepared and sterilized using a PES syringe filter with 0.22 μm pore size (MillexTM, MA). Sterile HBC-MA solution was added to the cell culture medium, and the final concentrations were 0.002, 0.02, 0.2, 1 and 2 mg/mL. Cells were inoculated in 100 μL of DMEM (10% FBS) supplemented with 50 U/mL penicillin at 10,000 cells per well in a 96-well plate and incubated for 24 hours at 37°C in 5% CO ₂ atmosphere, 100 μL HBC-MA was added in the given concentration of DMEM medium. After 24, 48 and 72 hours incubation, the WST-1 assay was tested. 10 μL of WST-1 was added to cells cultured in 100 mL of DMEM medium per well and incubated at 37°C for 4 hours. The absorbance of the sample was measured at 480 nm using a Microplate Spectrophotometer (Biotek Instruments, Inc., USA).

The measurement results are shown in Fig. 6, and the error bars represent the standard error (SE) of repeated measurements for each sample (n=6 per each group).

6, even at a high concentration of 2.0 mg/mL, the graph of the absorbance of NIH3T3 fibroblasts was significantly higher than that of the control group and is statistically significant (p<0.001). Cell viability showed about 28% higher absorbance compared to the absorbance of 2.0 mg/mL at 72 hours for the control without HBC-MA. In conclusion, HBC-MA did not show toxicity to NIH3T3 fibroblasts.

6. Scanning electron microscope analysis

The effect of the cross-linking method on the morphology of the HBC-MA hydrogel was observed by SEM. Hydrogel samples formed under different conditions were freeze-dried under liquid nitrogen to prevent structural collapse. After freeze drying for 24 hours, all samples were cut with a sharp knife to provide a clear view of the inside. The freeze-dried HBC-MA sample was fixed to a stub through carbon tape and coated with platinum. The cross-sectional shape was measured with a field emission scanning electron microscope. Morphology was investigated using a SU-8010 scanning electron microscope (Hitachi, Japan).

Figure 7a is a SEM image inside the temperature crosslinked HBC-MA hydrogel, Figure 7b is a SEM image inside the double crosslinked HBC-MA hydrogel, and Figure 7c is a 700 times magnified SEM inside the double crosslinked HBC-MA hydrogel The image is shown.

The SEM image clearly showed the difference in the pore size of the hydrogel according to the crosslinking method. All SEM samples were mixed with 3% HBC-MA. The pores observed by SEM are larger than the pore size of the hydrated hydrogel. However, samples lyophilized in the same way had the same effect on their structure. The temperature crosslinked hydrogel formed pores and was porous, but the pores were not small. On the other hand, after temperature crosslinking, the hydrogel having an additional photocrosslinking bond formed relatively more pores and formed pores of a small diameter to show excellent mechanical properties and induce a rigid network structure.

7. 4D printing possibility test

After completely dissolving HBC-MA (15%) prepared in the above example at 3% (W/V) in 0.05 M HCl, Eosin Y 0.1 mM, 1.5% triethanolamine (TEOA), 1% N-vinylcaprolactam (VC) was added to the HBC-MA solution. After the stirring was completed, the pH was adjusted to 7.4 using NaOH. The HBC-MA solution was sterilized using a PES syringe filter (MillexTM, MA) having a pore size of 0.22 μm. After completely removing the air bubbles, the prepared HBC-MA solution was transferred to a syringe and a 20G needle was prepared. The HBC-MA solution was always stored in a cooler place than room temperature. After sterilizing the third distilled water in an autoclave, it was maintained at 40°C and transferred to a Petri dish to create a sterilized underwater space. The HBC-MA solution was sprayed thinly using a syringe with a 20G needle. The compression force of the syringe was controlled by hand. The shape of the sample could be made freely, and the design was a mixture of curves and straight lines. Optical crosslinking was performed using Omnicure S2000 (Lumen Dynamics, Canada) equipped with a visible light filter (450 to 550 nm), and the light intensity was 35 mW/cm ² . The distance between the sample and the light source was maintained at 2.5 cm. The linear sample was exposed to light.

The process and results are shown in FIGS. 8A to 8D.

The solution contained in the syringe started to cause a temperature crosslinking reaction from the moment it was sprayed into water maintained at 40°C. The reaction took place very quickly. After about 10 seconds passed, the crosslinking reaction could be visually confirmed with an opaque color. The reactant became a hydrogel with moderate viscosity and elasticity (about 400 Pa) at a temperature similar to body temperature. In addition, a scaffold with pores was constructed internally. However, the cell adhesion rate of the hydrogel in which 3% HBC-MA was temperature-crosslinked was considerably decreased. Since the pore size is large, it is easy to inflow cells, but there is also a problem of outflow. It is believed that printing using only temperature crosslinking is difficult to support cells. In this experimental example, visible light was exposed here. It was observed using a contrast filter. The hydrogel exposed to visible light absorbed and emitted light even underwater. Hydrogel reacted rapidly to visible light even in water. In addition, there was no difficulty in crosslinking visible light even after temperature crosslinking. However, due to the limitation of the area of the light source, it was exposed to light while following a linear sample. The more light absorbed, the closer to yellow. It could be seen that the color was lightened throughout the sample. The photo-crosslinking reaction product became a photo-crosslinked hydrogel having elastic strength (20 to 50 kPa) in the visible light wavelength band, which has more safety than UV. The hydrogel with increased strength could be freely moved using tweezers. Chitosan hydrogel photo-crosslinked by methacrylate substitution improves cell adhesion.

Claims (13)

Hydrogel composition comprising a chitosan compound having a repeating unit represented by the following formulas 1 to 3:
[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,
R ₁ is hydrogen or an alkyl group having 1 to 4 carbon atoms,
R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

Is,
R ₅ is hydrogen or an alkyl group having 1 to 4 carbon atoms,
p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.20≤q≤0.50, 0.20≤r≤0.50, p>0 Satisfies the phosphorus condition.
The method of claim 1,
R ₁ is hydrogen, methyl or ethyl,
R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

Is,
R ₅ is hydrogen or a methyl group,
p, q, and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.20≤q≤0.30, 0.10≤r≤0.30, p>0 Hydrogel composition satisfying the phosphorus condition.
The method of claim 1,
R ₁ is hydrogen, methyl or ethyl,
R ₂ , R ₃ and R ₄ are each independently hydrogen, a methyl group, an ethyl group, or

Is,
R ₅ is hydrogen or a methyl group,
p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.30≤q≤0.40, 0.30≤r≤0.50, p>0 Hydrogel composition satisfying the phosphorus condition.
The method of claim 1,
The chitosan compound is a hydrogel composition comprising a repeating unit represented by the following Formula 4:
[Formula 4]

In Chemical Formula 4,
R ₆ are independently of each other hydrogen or

ego,
R ₇ is R ₅ is hydrogen or a methyl group.
The method of claim 1,
The chitosan compound is a hydrogel composition, characterized in that the repeating units represented by Chemical Formulas 1 to 3 have a crosslinked structure by either light or heat.
The method of claim 5,
Chitosan compound is a hydrogel composition maintaining a crosslinked structure at a temperature of 30 to 50 ℃.
The method of claim 5,
Chitosan compound is a hydrogel composition that maintains a crosslinked structure under light irradiation conditions having a wavelength of 300 to 800 nm.
A method for preparing a hydrogel composition comprising the step of preparing a chitosan compound having repeating units represented by the following formulas 1 to 3 from a mixture containing a chitosan solution, an epoxy compound, and a methacrylic anhydride:
[Formula 1]

[Formula 2]

[Formula 3]

In Formulas 1 to 3,
R ₁ is hydrogen or an alkyl group having 1 to 4 carbon atoms,
R ₂ , R ₃ and R ₄ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, or

Is,
R ₅ is hydrogen or an alkyl group having 1 to 4 carbon atoms,
p, q and r are the molar fractions of the repeating units represented by Formulas 1 to 3 contained in the chitosan compound, p+q+r=1, 0.20≤q≤0.50, 0.20≤r≤0.50, p>0 Satisfies the phosphorus condition.
The method of claim 8,
Methacrylic anhydride is a method for producing a hydrogel composition, characterized in that it is a compound represented by the following formula (5):
[Formula 5]

In formula 5,
R ₈ is hydrogen or an alkyl group having 1 to 4 carbon atoms.
The method of claim 8,
The epoxy compound is a method for producing a hydrogel composition, characterized in that the compound represented by the following formula (6):
[Formula 6]

In formula 6,
R ₉ is hydrogen or an alkyl group having 1 to 4 carbon atoms.
The method of claim 8,
The step of preparing the chitosan compound,
Reacting the chitosan solution with the epoxy compound to form an intermediate compound including the repeating units represented by Formulas 1 and 2; And
A method for producing a hydrogel composition comprising the step of reacting the intermediate compound with methacrylic anhydride to form a compound including a repeating unit represented by Formulas 1 to 3.
The method of claim 11,
Prior to the step of forming an intermediate compound,
A method for producing a hydrogel composition further comprising the step of adjusting the pH of the chitosan solution to 6 to 8 using a basic compound.
The method of claim 12,
The basic compound is ammonia (NH ₃ ), ammonium hydroxide (NH ₄ OH), magnesium hydroxide (Mg(OH)), potassium hydroxide (KOH), calcium hydroxide (Ca(OH) ₂ ), sodium hydroxide (NaOH), barium hydroxide (Ba(OH) ₂ ), aluminum hydroxide (Al(OH) ₃ ), iron hydroxide (Fe(OH) ₂ ), sodium hydrogen carbonate (NaHCO ₃ ), sodium carbonate (NaCO), calcium carbonate (CaCO ₃ ), potassium carbonate ( K ₂ CO ₃ ), methylamine (CH ₃ NH ₂ ) and aniline (C ₆ H ₅ NH ₂ ) The method of producing a hydrogel composition of at least one selected from the group consisting of.

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