JP6994147B2 - Cross-linking agent for high molecular weight polysaccharide or protein and its manufacturing method, cross-linked high molecular weight polysaccharide or cross-linking protein forming sol and its manufacturing method - Google Patents

Description

The present disclosure relates to a cross-linking agent for cross-linking a high molecular weight polysaccharide or a protein and a method for producing the same, a sol for forming a high molecular weight polysaccharide or a cross-linking protein and a method for producing the same.

In tissue regeneration engineering for the purpose of tissue reconstruction and functional regeneration, biomaterials are being developed all over the world to achieve these goals. For example, when an attempt is made to transplant a biopolymer polysaccharide or bioprotein chitosan, collagen, gelatin, etc. into a living body, the material itself cannot be molded due to poor mechanical strength, and when transplanted into a living body, it is immediately decomposed and absorbed. There is a problem that the stability in the living body is low, such as being damaged. Therefore, chemical cross-linking is known as one of the methods for enhancing the mechanical properties and in vivo stability of such high molecular weight polysaccharides or proteins.

As a conventional method of chemical cross-linking, Non-Patent Document 1 describes a method of chemical cross-linking using glutaraldehyde.

Further, Patent Document 1 describes a medical material obtained by cross-linking gelatin with succinimided poly-L-glutamic acid.

Non-Patent Document 2 describes water-soluble carbodiimide as a condensing agent for protein / peptide synthesis.

Non-Patent Document 3 describes a cross-linking agent called genipin, which is derived from gardenia and is orally taken as a Chinese herbal medicine.

Non-Patent Document 4 describes cytotoxicity when a diepoxy compound is used as a biomaterial.

Non-Patent Document 5 describes the effect of genipin cross-linking on collagen gel.

Patent Document 2 describes an aqueous collagen solution containing genipine and collagen, wherein the aqueous collagen solution has a gelation time of 30 minutes or more at 25 ° C.

Non-Patent Document 6 describes a collagen / genipin aqueous solution using pepsin-digested collagen (PSC) as a substrate.

Non-Patent Document 7 describes a method for polymerizing chitosan and genipin under basic conditions.

Japanese Unexamined Patent Publication No. 9-103479 Japanese Unexamined Patent Publication No. 2014-103985

Van Luin MJ. , Biomaterials, 13 (14), pp. 1017-1024 (1992) Huang Lee LL. , J. Biomed. , Mater. Res. , 24 (9), pp. 1185-1201 (1990) Change et al. Biomaterials 23, pp. 2447-2457 (2002) Nishi C. et al. , J Biomed Mater Res. 29 (7): 829-834 (1995) Harini G. Sundaragavan et al. , J Biomed Mater Res A. 87 (2), pp. 308-320 (2008) Yunoki S. , Et al. , International Journal of Biomaterials. 620765 (2013) MILF. , J Polymer Sci Part A 43, 1985-2000 (2005)

Although Non-Patent Document 1 and Patent Document 1 describe the use of glutaraldehyde as a cross-linking agent, Non-Patent Document 1 shows that glutaraldehyde is highly cytotoxic.
Further, Non-Patent Document 2 shows that when water-soluble carbodiimide is directly administered to cells, the cytotoxicity is high.
Genipin described in Non-Patent Document 3 is known to have extremely low cytotoxicity as compared with the above-mentioned glutaraldehyde, water-soluble carbodiimide, and the like. For example, it is known that the cytotoxicity is 5,000 to 10,000 times lower than that of glutaraldehyde (Non-Patent Document 4). However, it has been shown that even this genipin becomes more cytotoxic when the added concentration is increased (Non-Patent Document 5).

Regarding the above-mentioned genipin, according to Non-Patent Document 6, the gelling characteristics of a collagen / genipin mixed aqueous solution were clarified using collagen derived from pig skin that became telopeptide-free by pepsin digestion. At this time, it has been shown that the collagen / genipin mixed aqueous solution rapidly gels at body temperature while having room temperature fluidity of 30 minutes or more.
However, the present inventors have found that the use of genipin may not be suitable for applications where a shorter gelling time is required (for example, a hemostatic agent).
Further, Non-Patent Document 7 reports that genipin undergoes ring-opening polymerization by aldol condensation by OH protons of a strong alkaline aqueous solution.

As a result of diligent studies, the present inventors have found that the compound represented by the formula A described later has a very excellent cross-linking activity as a high molecular weight polysaccharide or a cross-linking agent for protein cross-linking, and that the formula A indicates that the compound has very excellent cross-linking activity. It has been found that the crosslinked polymer polysaccharide or crosslinked protein-forming sol containing the represented compound and the polymer polysaccharide or protein has a short gelation time.
By using a cross-linking agent having excellent cross-linking activity, cross-linking of high molecular weight polysaccharides or proteins can be performed even when used at a low concentration (for example, 1 mM to 6 mM) that suppresses toxicity to the living body. I think it is very useful in that it is possible.
In addition, since the gelation time of the crosslinked polymer polysaccharide or the sol for forming a crosslinked protein is short, the crosslinked polymer is also used in applications such as tissue adhesives and hemostatic agents, which require a short gelation time. We believe that it is very useful in that it makes it possible to apply sol for forming saccharides or crosslinked proteins.
Furthermore, the present inventors obtain the compound represented by the formula A as a compound in which the pyran ring structure portion of genipin is opened by heating, for example, genipin, but the crosslink height is increased by changing the heating conditions of genipin. It has been found that the gelation time of molecular polysaccharides or cross-linked protein-forming sol can be adjusted.
By adjusting the gelation time of the cross-linked polymer polysaccharide or the sol for forming the cross-linked protein, it is possible to provide a cross-linked protein that is required to have high fluidity at room temperature, for example, an endoscopic gel, and an adhesive for tissues. It is considered to be very useful because it enables the provision of materials for different purposes such as the provision of crosslinked polymer polysaccharides or crosslinked proteins that are required to have a short gelation time, such as blood-stopping agents. ..

The detailed mechanism by which the above effect is obtained is unknown, but since the rate of ring opening of genipin increases by heating, the probability of contact between genipin whose ring-opened pyran ring structure is opened and the amino group of the protein increases, and the protein It is speculated that it will be possible to quickly crosslink the amino group of.

An object to be solved by one embodiment of the present invention is to provide a high molecular weight polysaccharide having excellent cross-linking activity or a cross-linking agent for protein cross-linking.
An object to be solved by another embodiment of the present invention is to provide a crosslinked polymer polysaccharide or a crosslinked protein forming sol having a short gelation time.
An object to be solved by another embodiment of the present invention is to provide a method for producing a high molecular weight polysaccharide or a cross-linking agent for protein cross-linking having excellent cross-linking activity.
An object to be solved by another embodiment of the present invention is to provide a method for producing a crosslinked polymer polysaccharide or a sol for forming a crosslinked protein having a short gelation time.

The means for solving the above problems include the following aspects.
<1> A cross-linking agent for high molecular weight polysaccharide or protein cross-linking, which comprises a compound represented by the following formula A.

In the formula A, M represents a counter cation, R represents a substituent, and n represents an integer of 0 to 2.
<2> A cross-linking agent for high molecular weight polysaccharides or proteins, which is a reaction product obtained by heating genipin.
<3> A cross-linked polymer polysaccharide or cross-linked protein-forming sol containing the polymer polysaccharide or cross-linking agent for protein cross-linking according to <1> or <2>.
<4> The crosslinked polymer polysaccharide or crosslinked protein-forming sol according to <3>, wherein the polymer polysaccharide or protein is chitosan.
<5> The content of the cross-linked polymer polysaccharide or the cross-linking agent for protein cross-linking is 1 mmol / L to 6 mmol / L with respect to the total amount of the cross-linked polymer polysaccharide or the cross-linked protein-forming sol. The sol for forming a crosslinked polymer polysaccharide or a crosslinked protein according to <4>.
<6> The content of the polymer polysaccharide or protein is 0.5% by mass to 10% by mass with respect to the total mass of the crosslinked polymer polysaccharide or crosslinked protein-forming sol. 5> The crosslinked polymer polysaccharide or crosslinked protein-forming sol according to any one of 5>.
<7> The crosslinked polymer polysaccharide or crosslinked protein-forming sol according to any one of <3> to <6>, wherein the pH is 6.0 to 9.0.
<8> A method for producing a cross-linking agent for high molecular weight polysaccharide or protein cross-linking, which comprises a step of heating genipin in a phosphate buffer solution.
<9> The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to <8>, wherein the phosphate buffer solution is a sodium phosphate buffer solution.
<10> The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to <8>, wherein the phosphate buffer is a phosphate buffered saline.
<11> The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to <8>, wherein the phosphate buffer solution is a sodium dihydrogen phosphate solution.
<12> The polymer according to any one of <8> to <11>, wherein the heating temperature in the heating step is 20 ° C. to 70 ° C. and the heating time is 3 hours to 168 hours. A method for producing a cross-linking agent for cross-linking polysaccharides or proteins.
<13> For cross-linking polymer polysaccharide or cross-linking protein formation, which comprises a step of mixing the polymer polysaccharide or protein cross-linking agent according to <1> or <2> with the polymer polysaccharide or protein. How to make a sol.
<14> The polymer polysaccharide or the cross-linking agent for protein cross-linking obtained by the method for producing the cross-linking agent for polymer polysaccharide or protein according to any one of <8> to <12>, and the polymer. A method for producing a sol for forming a crosslinked polymer polysaccharide or a crosslinked protein, which comprises a step of mixing the polysaccharide or the protein.

According to one embodiment of the present invention, it is possible to provide a high molecular weight polysaccharide having excellent cross-linking activity or a cross-linking agent for protein cross-linking.
According to another embodiment of the present invention, it is possible to provide a crosslinked polymer polysaccharide having a short gelation time or a sol for forming a crosslinked protein.
According to another embodiment of the present invention, it is possible to provide a method for producing a high molecular weight polysaccharide or a cross-linking agent for protein cross-linking having excellent cross-linking activity.
According to another embodiment of the present invention, it is possible to provide a method for producing a crosslinked polymer polysaccharide or a sol for forming a crosslinked protein having a short gelation time.

It is the result of the spectrophotometer experiment in Examples 1, 2, 3 and Comparative Example 1. It is the result of having measured the storage viscoelasticity (G') in Example 4, Comparative Examples 2 and 3. It is a gelation curve of the sol for forming a crosslinked polymer polysaccharide using the genipin reaction products having different heating times. It is a graph which shows the relationship between the heating time of genipin and the gelation time of the sol for forming a crosslinked polymer polysaccharide. It is a graph which shows the evaluation result of the hardness of a gel. It is a graph which shows the dynamic viscoelasticity measurement result at the time of changing the concentration of a genipin reaction product. It is a chart which shows the measurement result of the UV absorption spectrum. It is a chart which shows the measurement result of the FT-IR spectrum. ¹³ It is a chart which shows the measurement result of 13 C-NMR spectrum.

Hereinafter, the present disclosure will be described in detail.
In the present disclosure, the description of "xx to yy" represents a numerical range including xx and yy.
Further, in the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.
In the present disclosure, with respect to the notation of a group in the compound represented by the formula, unless substituted or unsubstituted, or if the above group can further have a substituent, unless otherwise specified, it is not specified. It includes not only an unsubstituted group but also a group having a substituent. For example, if there is a description that "R represents an alkyl group" in the formula, it means "R represents an unsubstituted alkyl group or an alkyl group having a substituent".
In the present specification, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes.
Hereinafter, the present disclosure will be described in detail.

(Polysaccharide or cross-linking agent for protein cross-linking)
The first aspect of the high molecular weight polysaccharide or the cross-linking agent for protein cross-linking (hereinafter, also simply referred to as “cross-linking agent”) according to the present disclosure includes a compound represented by the following formula A.
The structure represented by the formula A may be a structure derived from genipin or a structure derived from another compound, but a structure derived from genipin is preferable.
The second aspect of the cross-linking agent according to the present disclosure is a reaction product obtained by heating genipin. It is presumed that the compound represented by the formula A, that is, the ring-opening reaction product of genipin is obtained by heating the genipin, but it is possible that the genipin reaction product having another structure is further contained. And so on, I'm not sure.
The polymer polysaccharide or protein cross-linking agent according to the present disclosure can crosslink a polymer polysaccharide or protein containing an amino group.
The cross-linking agent for protein cross-linking according to the present disclosure is considered to cross-link amino groups in high molecular weight polysaccharides or amino groups in proteins (for example, side chain amino groups of lysine (lysine)).

Preferred examples of R include an alkyl group, an alkoxy group, a halogen atom, and a hydroxy group.
n is preferably 0 or 1, more preferably 0, where M represents a counter cation. M may be bound to O ⁻ in the formula A or dissociated, but it is preferable that at least a part thereof is dissociated.
Further, the M is not particularly limited, and examples thereof include hydrogen ion, sodium ion, magnesium ion and the like.

(Manufacturing method of high molecular weight polysaccharide or cross-linking agent for protein cross-linking)
The method for producing a polymer polysaccharide or a cross-linking agent for protein cross-linking according to the present disclosure preferably includes a step of heating genipin in a phosphate buffer solution (hereinafter, also referred to as a “heating step”).
The high molecular weight polysaccharide or the cross-linking agent for protein cross-linking according to the present disclosure has an advantage that it can be easily applied to a living body, for example, it can be directly applied to the body by heating genipin in a phosphate buffer solution.

<Heating process>
[Phosphate buffer]
As the phosphate buffer used in the heating step, a known phosphate buffer can be used without particular limitation, but from the viewpoint of easy application to a living body, a sodium phosphate buffer is preferable, and phosphorus is preferable. It is more preferable to use acid buffered saline.
Further, from the viewpoint of facilitating preparation, a sodium dihydrogen phosphate solution is preferable.
As the phosphate buffer solution, for example, a 50 mmol / L disodium hydrogen phosphate aqueous solution (containing 140 mmol / L sodium chloride) and a 50 mmol / L sodium dihydrogen phosphate aqueous solution (containing 140 mmol / L sodium chloride) are contained. ) Can be adjusted as a pH 7.0, 140 mmol / L sodium chloride-containing 50 mmol / L phosphate buffer solution by stirring and mixing while measuring the pH with an pH meter using ultrapure water as a solvent. ..

[Heating method]
The heating method in the heating step is not particularly limited, but genipin is dissolved in a phosphate buffer solution having a pH of 6.0 to 9.0 at 0.5 mmol / L to 20 mmol / L to bring the temperature to 37 ° C. It is heated by.
By adjusting the heating temperature and heating time, the ratio of genipin to open the ring (ring-opening ratio) is adjusted.
The heating temperature is not particularly limited, but is preferably 20 ° C to 70 ° C, more preferably 30 ° C to 40 ° C.
The heating time is not particularly limited, but is preferably 3 hours to 168 hours, more preferably 3 hours to 72 hours, and even more preferably 6 hours to 48 hours.

(Cross-linked polymer polysaccharide or sol for forming cross-linked protein)
The cross-linked polymer polysaccharide or cross-linked protein-forming sol according to the present disclosure includes the polymer polysaccharide or the cross-linking agent for protein cross-linking according to the present disclosure, and the polymer polysaccharide or protein.
The crosslinked polymer polysaccharide or crosslinked protein-forming sol according to the present disclosure is heated to, for example, 37 ° C. to form crosslinks between the above-mentioned polymer polysaccharides or proteins by the cross-linking agent for protein cross-linking according to the present disclosure. , Gels are formed.

<Contents of high molecular weight polysaccharides or cross-linking agents for protein cross-linking>
The cross-linked polymer polysaccharide or the cross-linked protein-forming sol according to the present disclosure has a content of the cross-linked polymer polysaccharide or the cross-linking agent for protein cross-linking from the viewpoint of toxicity to a living body and gelation rate. Alternatively, it is preferably 1 mmol / L to 6 mmol / L, more preferably 0.5 mmol / L to 3 mmol / L, based on the total amount of the crosslinked protein-forming sol.

<Polysaccharide or protein>
The polymer polysaccharide or protein is not particularly limited, and known polymer polysaccharides or proteins are used.
It is very useful that the high molecular weight polysaccharide or the cross-linking agent for protein cross-linking according to the present disclosure can cross-link biopolymer polysaccharides or bioproteins such as chitosan, collagen and gelatin.
Therefore, as the protein, a biopolymer polysaccharide such as chitosan, collagen, gelatin or a bioprotein or a bioprotein is preferable, and chitosan is more preferable.

<Contents of high molecular weight polysaccharides or proteins>
The crosslinked polymer polysaccharide or the sol for forming a crosslinked protein according to the present disclosure may contain the polymer polysaccharide or protein alone or in combination of two or more.
The content of the cross-linked polymer polysaccharide or the protein in the cross-linked polymer polysaccharide or the sol for forming the cross-linked protein according to the present disclosure is the cross-linked polymer polysaccharide or the cross-linked protein from the viewpoint of improving the gel strength and shortening the gelation time. It is preferably 0.5% by mass to 10% by mass, more preferably 0.5% by mass to 5% by mass, and even more preferably 0.5% by mass to 3% by mass, based on the total mass of the forming sol.

<pH>
The pH of the crosslinked polymer polysaccharide or the sol for forming a crosslinked protein is preferably 6.0 to 9.0, and more preferably 7.0 to 8.0.
The pH is measured by a pH meter (NaVih F-71, manufactured by HORIBA, Ltd.).

(Method for producing cross-linked polymer polysaccharide or cross-linked protein forming sol)
The first aspect of the method for producing a crosslinked polymer polysaccharide or a sol for forming a crosslinked protein according to the present disclosure is a step of mixing a polymer polysaccharide or a crosslinking agent for protein cross-linking with a polymer polysaccharide or a protein. include.
The second aspect of the method for producing a crosslinked polymer polysaccharide or a sol for forming a crosslinked protein according to the present disclosure is a polymer polysaccharide obtained by the method for producing a crosslinked polymer polysaccharide or a crosslinked agent for protein crosslink according to the present disclosure. Alternatively, it comprises a step of mixing a cross-linking agent for cross-linking a protein with a polymer polysaccharide or a protein.
In any aspect, the mixing method is not particularly limited, and a known mixing method can be used.

Hereinafter, the present disclosure will be described in detail by way of examples, but the present disclosure is not limited thereto.

(Examples 1 to 3 and Comparative Example 1)
<Preparation of chitosan solution>
3 g chitosan powder (manufactured by Wako Pure Chemical Industries, Ltd.) was placed in a 400 mmol / L acetic acid solution (300 mL) and dissolved to prepare a 1% chitosan solution. After dispensing the 1% chitosan solution into the dialysis tube, the dialysis tube was immersed in ultrapure water for desalting. The ultrapure water was replaced every 3 hours, and this work was carried out 3 times. Then, it was dispensed into a 15 mL tube and stored in a refrigerator (4 ° C.).

<Preparation of phosphate buffer (NPB)>
An aqueous solution of disodium hydrogen phosphate (containing 140 mmol / L sodium chloride) having a concentration of 50 mmol / L and an aqueous solution of sodium dihydrogen phosphate having a concentration of 50 mmol / L (containing 140 mmol / L sodium chloride) were prepared using ultrapure water as a solvent. did. While measuring the pH with a pH meter (manufactured by HORIBA, Ltd., trade name "NAVIh F-71"), these are stirred and mixed to adjust pH 7.0, 140 mmol / L sodium chloride-containing 50 mmol / L phosphate buffer. This was designated as NPB.

<Preparation of genipin solution>
Genipin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in NPB to prepare a genipin aqueous solution having a concentration of 2 mmol / L to 10 mmol / L. This was placed in a 50 mL tube and then stored in a refrigerator (4 ° C.).

<Heating of genipin solution>
The genipin solution was heated in a 37 ° C. water bath for 6 hours, 12 hours or 24 hours. Then, a 0.45 μm size filter (Merc millipore product name Filter unit) was attached to the tip of a 10 mL syringe (Terumo Syringe SS-10LZP), and then the heated genipin was filtered. After filtering with a filter, it was dispensed into a 50 mL tube and stored in a refrigerator as a genipin solution. The 6-hour heated genipin reaction solution is Ge (6), the 12-hour heated genipin reaction solution is Ge (12), the 24-hour heated genipin reaction solution is Ge (24), and the unheated genipin solution is used. It was set to Ge (0).

< ¹³ C-NMR experiment>
Structural changes were evaluated by ¹³ C-NMR (JEOL product name JNM-ECA600) using Ge (24).
Ge (24) was frozen and then dried in a freeze dryer for 24 hours.
_The lyophilized sample was dissolved in a D2O solution and then injected into an NMR tube (diameter 4 mm). After that, the frequency of ^13C was 150 MHz.

<Spectrophotometer experiment>
The maximum absorption wavelength of the genipin solutions Ge (6), Ge (12), Ge (24) and Ge (0) was examined using a spectrophotometer (UV-3100S manufactured by Shimadzu Corporation). The measurement wavelength was 200 to 400 nm.

<Dynamic viscoelasticity experiment>
Using chitosan as a substrate, the cross-linking activity of the genipin reactant solutions Ge (6), Ge (12), Ge (24) and Ge (0) was evaluated by dynamic viscoelasticity measurement. As the dynamic viscoelastic device, the trade name "HAAKE MARS III" manufactured by Thermo Fisher Scientific Co., Ltd. was used. Geometry and measurement program were used properly according to the purpose of the experiment. The measurement conditions are shown below.

Table 1 below shows the chitosan content used in each Example and Comparative Example, the genipin reaction product (genipin) content of the cross-linking agent for high molecular weight polysaccharides or proteins, the solvent, the heating time, and the type of the genipin reaction product solution. Described.

(Result of Example 1)
The results of the spectrophotometer experiment using the genipin reactant solution of Example 1 in Table 1 are shown in FIG. As a result of Example 1, it can be seen that the maximum absorption wavelength is 250 nm, and the maximum absorption wavelength is shifted as compared with the maximum absorption wavelength of 240 nm of the genipin solution of Comparative Example 1 which has not been heated.
It is presumed that this is because the maximum absorption wavelength has shifted due to the heating of genipin.

(Result of Example 2)
The results of the spectrophotometer experiment using the genipin reactant solution of Example 2 in Table 1 are shown in FIG. As a result of Example 2, the maximum absorption wavelength is 248 nm, and the maximum absorption wavelength is shifted as compared with the maximum absorption wavelength of 240 nm of the Genipin solution of Comparative Example 1 and the maximum absorption wavelength of 246 nm of the Genipin solution of Example 1. You can see that.
It is presumed that this is because the maximum absorption wavelength was shifted due to the ring opening of genipin, and it is presumed that the genipin reaction product solution in Example 2 had more genipin opened than the genipin solution in Example 1. Will be done.

(Result of Example 3)
The results of the spectrophotometer experiment using the genipin reactant solution of Example 3 in Table 1 are shown in FIG. As a result of Example 3, the maximum absorption wavelength was 252 nm, the maximum absorption wavelength of the genipine solution of Comparative Example 1 was 240 nm, the maximum absorption wavelength of the genipine reaction product solution of Example 1 was 246 nm, and the maximum absorption wavelength of the genipine reaction product solution of Example 2. It was found that the maximum absorption wavelength was shifted as compared with the absorption wavelength of 248 nm.
It is presumed that this is because the maximum absorption wavelength was shifted due to the opening of the ring of genipin, and the solution of the genipin reactant in Example 3 was further opened than the solution of the genipin reactant in Examples 1 and 2. It is presumed that there are many genipins.

(Result of Comparative Example 1)
The results of the spectrophotometer experiment using the genipin solution of Comparative Example 1 in Table 1 are shown in FIG. As a result of Comparative Example 1, the maximum absorption wavelength was 240 nm.

(Example 4 and Comparative Examples 2 to 3)
<Preparation of gelatin solution>
Gelatin powder (MP Biomedicals) was prepared with ultrapure water so as to have a concentration of 10% by mass, and then dissolved in a water bath at 70 ° C. This was divided into small portions in a 15 mL centrifuge tube and stored in a refrigerator (4 ° C.).

[Crosslinking activity at 37 ° C]
The above-mentioned gelatin solution was mixed with the above-mentioned Ge (24) or Ge (0), and the measurement was started through a stirring operation.
After holding at 23 ° C. for 60 seconds, the temperature was raised to 37 ° C. for 30 seconds, and the stored viscoelasticity was measured when the temperature was kept constant at that temperature for 30 minutes. The measurement conditions were as follows.
Geometry: Parallel sensor (inner diameter 60 mm)
Measurement profile: Oscillation measurement by CD (Control Deformation) mode (shear stress = 1 Pa, temperature 23 ° C. to 37 ° C., frequency = 1 Hz, time = 60 minutes).
The time from the start of the measurement until the storage viscoelasticity (G') and the loss elastic modulus (G'') intersect was defined as the “gelling time at 37 ° C.”.

Table 2 below shows the gelatin content used in each Example and Comparative Example, the genipin reaction product (genipin) content of the high molecular weight polysaccharide or the cross-linking agent for protein cross-linking, the solvent, the heating time, and the type of the genipin reaction product solution. Described.

(Results of Example 4 and Comparative Examples 2 to 3)
The results of measuring the stored viscoelasticity (G') using the crosslinked polymer polysaccharides or crosslinked protein-forming sol of Example 4 of Table 2 or Comparative Examples 2 to 3 are shown in FIG. The measurement temperature was 37 ° C.
From the results shown in FIG. 2, the gelation time is shorter when heated genipin is used as compared with the case where unheated genipin is used, even when gelatin is used as a substrate. You can see that.

(Examples 5 to 10 and Comparative Examples 4 to 5)

<Material>
Details of the compounds used in the following examples are as follows.
・ Genipin powder: manufactured by Wako Pure Chemical Industries, Ltd.
・ Chitosan 100: manufactured by Wako Pure Chemical Industries, Ltd. ・ Acetic acid: manufactured by Wako Pure Chemical Industries, Ltd. ・ Sodium dihydrogen phosphate: manufactured by Wako Pure Chemical Industries, Ltd. ・ Disodium hydrogen phosphate: manufactured by Wako Pure Chemical Industries, Ltd. Made by Co., Ltd. ・ Heavy water: Made by ISOTEC Co., Ltd.

<Preparation of phosphate buffer>
An aqueous solution of disodium hydrogen phosphate and an aqueous solution of sodium dihydrogen phosphate were prepared at a concentration of 100 mmol / L and mixed so that the pH became 7.0 (PB-100). A phosphate buffer solution having a concentration of 400 mM was also prepared by the same operation (PB-400).

<Preparation of chitosan aqueous solution>
2 g of a powder of chitosan 100 was added to a 5% aqueous acetic acid solution, and the mixture was stirred until the chitosan was completely dissolved. The obtained aqueous solution was placed in a dialysis tube (molecular weight cut off: 1 kDa) and dialyzed against pure water. After confirming that the pH of the dialysis external solution exceeded 5.0, the chitosan aqueous solution, which was the dialysis external solution, was recovered. A part thereof was taken out and dried at 60 ° C. overnight, and the chitosan concentration was determined from the mass. Ion-exchanged water was added to adjust the concentration to 1.4% by mass, which was used in the following experiments.

<Preparation of genipin reaction product solution>
Genipin powder (5 mg / mL) was added to 200 mL of PB-100 or ion-exchanged water and completely dissolved in a magnetic stirrer at room temperature. Ge dissolved in PB-100 was heated in a water bath at 37 ° C. to prepare a genipin reaction product solution having a heating time of 12 hours, 24 hours, 36 hours, and 48 hours.
Ge-12 for a genipine reactant solution with a heating time of 12 hours, Ge-24 for a genipine reactant solution with a heating time of 24 hours, Ge-36 for a genipine reactant solution with a heating time of 36 hours, and 48 hours for a heating time. The genipine reaction product solution was designated as Ge-48.
In addition, a genipin solution dissolved in ion-exchanged water was mixed with PB-400 immediately before use and diluted, and a genipin solution using PB-100 as a solvent was defined as Ge-0.

<Manufacturing of cross-linked polymer polysaccharide forming sol>
A predetermined weight of a 1.4 mass% chitosan aqueous solution was added to a biological tube (capacity 15 mL or 50 mL), and a magnetic stirrer was loaded to promote stirring. The above Ge-12, Ge-24, Ge-36, Ge-48, and Ge-0 diluted with PB-100 to 50 μg / mL were added in equal volumes with the specific gravity of the chitosan aqueous solution as 1. After shaking the tube, the mixture was centrifuged at 2500 rpm for 30 seconds to defoam, and a sol for forming a crosslinked polymer polysaccharide was produced. After production, it was immediately subjected to dynamic viscoelasticity measurement.

<Evaluation of genipin cross-linking characteristics by dynamic viscoelasticity measurement>
[Tracking changes in viscoelasticity]
A rheometer (HAAKE Mars III, manufactured by Thermo fisher Scientific Inc.) was used to measure the dynamic viscoelasticity of the sol for forming crosslinked polymer polysaccharides, and the time change of viscoelasticity was tracked. A sol for forming crosslinked polymer polysaccharides is poured on the bottom of a parallel plate sensor (diameter 60 mm) whose temperature is set to 37 ° C., the upper sensor is brought close to a gap of 1 mm, and dynamic viscoelasticity at a frequency of 1 Hz under constant stress (1 Pa). The measurement was started. Changes in the storage modulus (G') and loss modulus (G'') were recorded, and the time to reach the intersection (gel point) of G'and G'' was defined as the gelation time.

[Evaluation of gel hardness]
The dynamic viscoelasticity of the gel prepared on the petri dish was measured, and the cross-linking ability of the genipin reactant was evaluated from the hardness of the cross-linked chitosan gel. The cross-linked polymer polysaccharide-forming sol was poured into a biological dish having an inner diameter of 35 mm and allowed to stand in a humidified 37 ° C. incubator to prevent the gel from drying. After 24 hours, the dish was removed from the incubator and subjected to dynamic viscoelasticity measurement. A silicone rubber sheet was inserted into the bottom of the dish to prevent slipping, and sandpaper was attached to the upper sensor with an inner diameter of 20 mm in advance. The upper sensor was brought into contact with the gel, and stress dispersion measurement (0.1 Pa to 100 Pa) at a frequency of 0.2 Hz was performed. G'at stress 1 Pa or 10 Pa for which linear viscoelasticity measurements were performed was recorded.

The details of the crosslinked polymer polysaccharide-forming sol used in each Example or Comparative Example in the tracking of the viscoelastic change and the evaluation of the hardness of the gel are as shown in Table 3.
In Example 9, Example 10 and Comparative Example 5, the genipin solution was diluted and then mixed with the chitosan aqueous solution so that the content (final concentration) of the genipin reactant or genipin was as follows.

The results of tracking the change in viscoelasticity and evaluating the hardness of the gel are shown in FIGS. 3 to 6.
FIG. 3 is a gelation curve of a sol for forming a crosslinked polymer polysaccharide using genipin reactants having different heating times.
From FIG. 3, it can be seen that the increase in G'after 10 minutes of the cross-linked polymer polysaccharide-forming sol is promoted as the heating time of genipin becomes longer.
When Ge-0 was used, G'showed the most moderate increase, and G'10 minutes after the start of measurement was 0.65 Pa. On the other hand, when Ge-48 was used, G'showed the sharpest increase, and G'at 10 minutes after the start of measurement reached 41 Pa.

FIG. 4 shows the relationship between the heating time of genipin and the gelation time of the sol for forming crosslinked polymer polysaccharides. The longer the heating time, the shorter the gelation time, reflecting the relationship between the heating time of genipin and G'shown in FIG. The gelation time was 5.8 minutes when Ge-0 was used, whereas the gelation time was shortened to 0.6 minutes with Ge-48.

In the evaluation of the hardness of the gel, G'after 24 hours of the crosslinked polymer polysaccharide forming sol prepared on the dish is shown in FIG. The elastic modulus of the gel decreased as the heating time of genipin increased. The gel elastic modulus was 1999 ± 173 Pa when Ge-0 was used, but decreased to 726 ± 74 Pa when Ge-48 was used.

FIG. 6 shows the results of dynamic viscoelasticity measurement when the concentration of the genipin reactant was changed. It can be seen that at the same Ge concentration (500 μg / ml), gelation with Ge-24 is faster than with Ge-0. When the concentration of Ge-24 was reduced to 75 μg / ml, the gelation rate became a value close to Ge-0 of 500 μg / ml.

As described above, it can be seen that by using the heated genipin according to this example, a high molecular weight polysaccharide having excellent cross-linking activity or a cross-linking agent for protein cross-linking can be provided.
Here, excellent cross-linking activity is also useful in that the cross-linking rate increases when added at the same concentration, and the concentration of the cross-linking agent for equalizing the cross-linking rate is set to a low concentration. In terms of being able to do so, it is considered to be very useful in consideration of cytotoxicity and the like.
Further, according to this embodiment, it can be seen that the gelation time (gelation rate) can be adjusted by adjusting the heating time of genipin.

[Structural analysis of genipin]
Ultraviolet (UV) spectroscopic analysis, Fourier transform infrared (FT-IR) spectroscopic analysis, and ^13C nuclear magnetic resonance (NMR) analysis were performed to analyze the structural changes of genipine due to heating.

-UV spectroscopic analysis-
Using a spectrophotometer (UV-3100S, manufactured by Shimadzu Corporation), the UV spectra of Ge-0 (Comparative Example 6), Ge-24 (Example 11) and Ge-48 (Example 12) were measured at wavelengths of 200 to 300 nm. It was measured in the range of. The concentrations of Ge-0, Ge-24 and Ge-48 after preparation were too high for UV spectroscopic analysis, so PB-100 was added to dilute the concentration to 25 μg / mL before measurement.

-FT-IR-
The functional group of genipin was analyzed using an FT-IR device (FT / IR-6300, manufactured by JASCO Corporation). 10 mg of freeze-dried powder of Ge-0 (Comparative Example 7) or Ge-48 (Example 13) and 500 mg of potassium bromide powder are mixed, homogenized in a mortar, and then placed in a tablet molding machine for pressure tablet making. did. The measurement wavelength was from 3800 cm ^-1 to 400 cm ^-1 , the resolution was 4 cm ^-1 , and the number of integrations was 64 times. The measurement of the dried sample of PB-100 was performed at the same time as the blank measurement. The spectrum of each Ge was displayed as the difference spectrum between the measured spectrum and the spectrum of PB-100.

^-13 C-NMR-
Using an NMR device (JNM-ECA600, JEOL Resonance inc), ¹³ C-NMR quantitative measurement in a decoupling mode was performed. For Ge-0 (Comparative Example 8), Ge powder (5 mg / mL) was dissolved in deionized water, the Ge aqueous solution was concentrated using an evaporator (RE301B-V, manufactured by Yamato Kagaku), and PB-400 was added. The solvent concentration was adjusted to PB-100. Ge-48 (Example 14) also concentrated the aqueous solution using an evaporator. Then, 1/4 volume of heavy water was added and filled in an NMR tube (diameter 4 mm). The measurement was performed at a frequency of 150 MHz.

<Measurement result>
FIG. 7 shows the measurement results of the UV absorption spectrum. Ge-0 showed maximum absorption at a wavelength of 240 nm. On the other hand, the maximum absorption wavelength was shifted to the long wavelength side by heating the genipin, and the maximum absorption wavelength of Ge-48 was 252 nm. The UV absorption spectra of Ge-12, Ge-24, and Ge-36 almost overlapped with Ge-48.

FIG. 8 shows the measurement results of the FT-IR spectrum. Comparing Ge-0 and Ge-48, the peaks attributed to C = O of the dihydropyran ring (1632 cm ^-1 ) and the peaks attributed to the hydroxyl group of genipin (3285 cm ^-1 and 3378 cm ^-1 ) decreased at the same time. ..

FIG. 9 shows the measurement results of the ¹³ C-NMR spectrum. The attribution of the ¹³ C-NMR spectra of carbon constituting genipin is Michael F, Yiu-FAI NG, Paul D, “Mechanism and Kinetics of the Crosslinking Reaction between Biopolymers Containing Priamry Amine Groups and Genipin”, Marine Drugs, 2003: 8, Refer to 3941-3953, and the attribution of each carbon is shown in the figure. Two clear differences between Ge-0 and Ge-48 were observed. The first is that two distinct peaks appeared in Ge-48 in the carbonyl carbon region (180 to 210 ppm), which was not seen in Ge-0, and the second was that the number of peaks was Ge-0. This is a significant increase from 12 lines.

The shift in the maximum absorption wavelength of the UV spectrum (Fig. 7) means that a structural change has occurred in the aromatic ring of genipin that changes the state of π electrons.
A decrease in the absorption peak derived from the pyran ring in the FT-IR spectrum (FIG. 8) suggests cleavage of the pyran ring.
In the ¹³ C-NMR spectrum, at least two new peaks appeared in the carbonyl carbon region, which was not observed in Ge-0 (Fig. 9). Considering the decrease in the absorption peak intensity derived from the hydroxyl group in the FT-IR spectrum, the formation of aldol by cleavage of the pyran ring is strongly suggested, and it is considered that at least the compound represented by the formula A is contained.
The above analysis suggests that the pyran ring of genipin is opened by heating, but from the many peaks observed in the ¹³ C-NMR spectrum, it is considered that there are multiple patterns of structural change of genipin by heating.

Claims (10)

A cross-linking agent for high molecular weight polysaccharides or proteins, which comprises a compound represented by the following formula A.

In the formula A, M represents a counter cation, R represents a substituent, and n represents an integer of 0 to 2. A cross-linking agent for high molecular weight polysaccharides or proteins, which comprises a reactant obtained by heating genipin. The cross-linking agent for cross-linking a high molecular weight polysaccharide or protein according to claim 1 or 2, wherein the high molecular weight polysaccharide or protein is chitosan. A method for producing a high molecular weight polysaccharide or a cross-linking agent for protein cross-linking, which comprises a step of heating genipin in a phosphate buffer solution. The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to claim 4 , wherein the phosphate buffer solution is a sodium phosphate buffer solution. The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to claim 4 , wherein the phosphate buffer is a phosphate buffered saline. The method for producing a polymeric polysaccharide or a cross-linking agent for protein cross-linking according to claim 4 , wherein the phosphate buffer solution is a sodium dihydrogen phosphate solution. The high molecular weight polysaccharide or protein according to any one of claims 4 to 7 , wherein the heating temperature in the heating step is 20 ° C. to 70 ° C. and the heating time is 3 hours to 168 hours. A method for producing a cross-linking agent for cross-linking. Formation of a crosslinked polymer polysaccharide or a crosslinked protein comprising a step of mixing the polymer polysaccharide or the cross-linking agent for protein cross-linking according to any one of claims 1 to 3 with the polymer polysaccharide or protein. How to make sol for. The high molecular weight polysaccharide or the cross-linking agent for protein cross-linking obtained by the method for producing the high molecular weight polysaccharide or the cross-linking agent for protein cross-linking according to any one of claims 4 to 8 , and the high molecular weight polysaccharide or protein. A method for producing a crosslinked polymer polysaccharide or a sol for forming a crosslinked protein, which comprises a step of mixing.

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