JP6811956B2 - Decomposition controllable hydrogel - Google Patents
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- JP6811956B2 JP6811956B2 JP2016173797A JP2016173797A JP6811956B2 JP 6811956 B2 JP6811956 B2 JP 6811956B2 JP 2016173797 A JP2016173797 A JP 2016173797A JP 2016173797 A JP2016173797 A JP 2016173797A JP 6811956 B2 JP6811956 B2 JP 6811956B2
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- 239000000017 hydrogel Substances 0.000 title claims description 78
- 238000000354 decomposition reaction Methods 0.000 title claims description 39
- 229920000310 Alpha glucan Polymers 0.000 claims description 53
- 125000003277 amino group Chemical group 0.000 claims description 35
- 150000001875 compounds Chemical class 0.000 claims description 35
- 125000003172 aldehyde group Chemical group 0.000 claims description 26
- 230000015572 biosynthetic process Effects 0.000 claims description 18
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 18
- 235000010418 carrageenan Nutrition 0.000 claims description 17
- 239000000679 carrageenan Substances 0.000 claims description 17
- 229920001525 carrageenan Polymers 0.000 claims description 17
- 229940113118 carrageenan Drugs 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 15
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 15
- 239000003349 gelling agent Substances 0.000 claims description 12
- 230000003647 oxidation Effects 0.000 claims description 9
- 238000007254 oxidation reaction Methods 0.000 claims description 9
- 229920002307 Dextran Polymers 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 8
- 125000000217 alkyl group Chemical group 0.000 claims description 7
- 229920006295 polythiol Polymers 0.000 claims description 7
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 3
- 230000000737 periodic effect Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 description 36
- 239000000499 gel Substances 0.000 description 29
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 238000001879 gelation Methods 0.000 description 22
- 238000005259 measurement Methods 0.000 description 19
- 239000007864 aqueous solution Substances 0.000 description 15
- 238000005160 1H NMR spectroscopy Methods 0.000 description 14
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 14
- 239000004471 Glycine Substances 0.000 description 11
- 239000008186 active pharmaceutical agent Substances 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical class O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 description 7
- 238000005576 amination reaction Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 150000001299 aldehydes Chemical class 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 6
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 5
- 229960004308 acetylcysteine Drugs 0.000 description 5
- 239000000853 adhesive Substances 0.000 description 5
- 230000001070 adhesive effect Effects 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 150000004804 polysaccharides Chemical class 0.000 description 4
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- FZERHIULMFGESH-UHFFFAOYSA-N N-phenylacetamide Chemical compound CC(=O)NC1=CC=CC=C1 FZERHIULMFGESH-UHFFFAOYSA-N 0.000 description 2
- 150000004662 dithiols Chemical class 0.000 description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethanethiol Chemical compound CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 2
- -1 for example Chemical compound 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- ZTGQZSKPSJUEBU-UHFFFAOYSA-N 3-bromopropan-1-amine Chemical compound NCCCBr ZTGQZSKPSJUEBU-UHFFFAOYSA-N 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- 229960001413 acetanilide Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- SMTOKHQOVJRXLK-UHFFFAOYSA-N butane-1,4-dithiol Chemical compound SCCCCS SMTOKHQOVJRXLK-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 125000003055 glycidyl group Chemical group C(C1CO1)* 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- RPQRDASANLAFCM-UHFFFAOYSA-N oxiran-2-ylmethyl prop-2-enoate Chemical compound C=CC(=O)OCC1CO1 RPQRDASANLAFCM-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- NCNISYUOWMIOPI-UHFFFAOYSA-N propane-1,1-dithiol Chemical compound CCC(S)S NCNISYUOWMIOPI-UHFFFAOYSA-N 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
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- 230000007704 transition Effects 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
Description
本発明は、分解制御性ハイドロゲルに関する。 The present invention relates to a degradation controllable hydrogel.
医療用、特に外科手術用の接着剤として、分解性のハイドロゲル及びゲル化剤が、開発されている（特許文献１、特許文献２）。このような医療用接着剤は、生体組織の接着、充填、癒着防止、止血などに用いられる。そして、これらの医療用接着剤用のハイドロゲルは、目的を果たした後に、生体内で適切に分解されることが求められている。 Degradable hydrogels and gelling agents have been developed as adhesives for medical use, especially for surgery (Patent Documents 1 and 2). Such medical adhesives are used for adhesion, filling, adhesion prevention, hemostasis and the like of living tissues. Then, these hydrogels for medical adhesives are required to be appropriately decomposed in a living body after fulfilling the purpose.
さらに、このようなハイドロゲル及びゲル化剤は、ドラッグデリバリー用途の基材にも使用されており、例えば、ゼラチンなどのタンパク質性のゲル、ヒアルロン酸やアルギン酸などの多糖類性のゲルなどが知られている。これらのＤＤＳ基材用のハイドロゲルは、薬物放出の目的のために、生体内で適切に分解されることが求められている。 Furthermore, such hydrogels and gelling agents are also used as base materials for drug delivery applications, and for example, protein gels such as gelatin and polysaccharide gels such as hyaluronic acid and alginic acid are known. Has been done. These hydrogels for DDS substrates are required to be properly degraded in vivo for the purpose of drug release.
このように、生体内で適切に分解されるように、分解の制御が可能である、ハイドロゲルが求められていた。 As described above, a hydrogel capable of controlling the decomposition so as to be appropriately decomposed in the living body has been required.
したがって、本発明の目的は、分解制御性を備えた、新規なハイドロゲルを提供することにある。 Therefore, an object of the present invention is to provide a novel hydrogel having decomposition controllability.
本発明者は、分解制御性を備えたハイドロゲルについて、鋭意研究を行ってきた。特許文献１及び特許文献２に開示されたハイドロゲルは、非常に優れたハイドロゲルではあるが、ゲル化の直後からゲルの分解の反応が開始してしまうという制約を持っていることを踏まえて、さらに分解制御性に優れたハイドロゲルの開発を行ってきた。 The present inventor has been diligently researching a hydrogel having decomposition controllability. Although the hydrogels disclosed in Patent Documents 1 and 2 are very excellent hydrogels, they have a restriction that the reaction of gel decomposition starts immediately after gelation. In addition, we have been developing hydrogels with excellent decomposition controllability.
そして、後述するハイドロゲルによって、上記目的を達成できることを見いだして、本発明に到達した。 Then, they have found that the above object can be achieved by the hydrogel described later, and have reached the present invention.
したがって、本発明は次の（１）以下を含む。
（１）
重量平均分子量が２０００〜２０万の範囲にあるα−グルカンと、次の式Ｉ：
で表される化合物を反応させて、次の式ＩＩ：
で表される基を、α−グルカンへ導入する工程、
式ＩＩで表される基が導入されたα−グルカンを、過ヨウ素酸又は過ヨウ素酸塩で酸化して、アルデヒド基を、α−グルカンへ導入する工程、
を含む、式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入されてなる、ゲル化剤の製造方法。
（２）
（１）で製造された、式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入されてなる、ゲル化剤を、ポリチオール性還元剤によって架橋反応させて、ハイドロゲルを形成する工程、
を含む、ハイドロゲルの製造方法。
（３）
（２）で製造されたハイドロゲルへ、ハイドロゲルの形成前又はハイドロゲルの形成後に、アミノ基を有する化合物を添加して、ハイドロゲルを分解する方法。
（４）
（２）で製造されたハイドロゲルへ、ハイドロゲルの形成前又はハイドロゲルの形成後に、アミノ基を有する化合物を添加して、ハイドロゲルの分解を制御する方法。
（５）
重量平均分子量が２０００〜２０万の範囲にあるα−グルカンに対して、次の式ＩＩ：
で表される基が、α−グルカン中のＯＨ基のＨに置換されて、α−グルカンのグルコース単位あたり１０〜５０％の範囲の導入率で導入され、
過ヨウ素酸酸化によるアルデヒド基が、α−グルカンのグルコース単位あたり２５〜７５％の範囲の導入率で導入されてなる、修飾α−グルカン化合物。
（６）
重量平均分子量が２０００〜２０万の範囲にあるα−グルカンが、デキストランである、（５）に記載の修飾α−グルカン化合物。
（７）
（５）〜（６）の何れかに記載の修飾α−グルカン化合物が、ジチオスレイトールによって架橋されてなる、ハイドロゲル。
（８）
アミノ基を有する化合物がハイドロゲル中にさらに含まれている、（７）に記載のハイドロゲル。
（９）
アミノ基を有する化合物が、アミノ化κカラギーナンである、（８）に記載のハイドロゲル。
（１０）
（７）〜（９）の何れかに記載のハイドロゲルからなる、分解制御性ハイドロゲル。
（１１）
（５）〜（６）の何れかに記載の修飾α−グルカン化合物からなる、ゲル化剤。
Therefore, the present invention includes the following (1) and the following.
(1)
Α-Glucan having a weight average molecular weight in the range of 2000 to 200,000 and the following formula I:
By reacting the compound represented by, the following formula II:
The process of introducing the group represented by (1) into α-glucan,
A step of oxidizing an α-glucan into which a group represented by the formula II has been introduced with periodic acid or a periodate to introduce an aldehyde group into the α-glucan.
A method for producing a gelling agent, which comprises introducing a group represented by the formula II and an aldehyde group into α-glucan.
(2)
A step of forming a hydrogel by cross-linking a gelling agent produced in (1), which comprises introducing a group represented by the formula II and an aldehyde group into α-glucan, with a polythiol reducing agent.
A method for producing a hydrogel, including.
(3)
A method for decomposing a hydrogel by adding a compound having an amino group to the hydrogel produced in (2) before or after the formation of the hydrogel.
(4)
A method of controlling the decomposition of a hydrogel by adding a compound having an amino group to the hydrogel produced in (2) before or after the formation of the hydrogel.
(5)
For α-glucans with a weight average molecular weight in the range of 2000-200,000, the following formula II:
The group represented by is substituted with H of the OH group in α-glucan and introduced at an introduction rate in the range of 10 to 50% per glucose unit of α-glucan.
A modified α-glucan compound in which an aldehyde group due to oxidation with periodate is introduced at an introduction rate in the range of 25 to 75% per glucose unit of α-glucan.
(6)
The modified α-glucan compound according to (5), wherein the α-glucan having a weight average molecular weight in the range of 2000 to 200,000 is dextran.
(7)
A hydrogel obtained by cross-linking the modified α-glucan compound according to any one of (5) to (6) with dithiothreitol.
(8)
The hydrogel according to (7), wherein the compound having an amino group is further contained in the hydrogel.
(9)
The hydrogel according to (8), wherein the compound having an amino group is aminated reductive carrageenan.
(10)
A decomposition controllable hydrogel comprising the hydrogel according to any one of (7) to (9).
(11)
A gelling agent comprising the modified α-glucan compound according to any one of (5) to (6).
本発明によれば、分解制御性に優れたハイドロゲルを得ることができる。 According to the present invention, a hydrogel having excellent decomposition controllability can be obtained.
具体的な実施の形態をあげて、以下に本発明を詳細に説明する。本発明は、以下にあげる具体的な実施の形態に限定されるものではない。 The present invention will be described in detail below with reference to specific embodiments. The present invention is not limited to the specific embodiments listed below.
［修飾α−グルカン化合物（ゲル化剤）の製造］
本発明によるゲル化剤（修飾α−グルカン化合物）は、重量平均分子量が２０００〜２０万の範囲にあるα−グルカンと、次の式Ｉ：
で表される化合物を反応させて、次の式ＩＩ：
で表される基を、α−グルカンへ導入する工程、
式ＩＩで表される基が導入されたα−グルカンを、過ヨウ素酸又は過ヨウ素酸塩で酸化して、アルデヒド基を、α−グルカンへ導入する工程、を含む方法によって、製造することができる。
[Manufacturing of modified α-glucan compound (gelling agent)]
The gelling agent (modified α-glucan compound) according to the present invention includes α-glucan having a weight average molecular weight in the range of 2000 to 200,000 and the following formula I:
By reacting the compound represented by, the following formula II:
The process of introducing the group represented by (1) into α-glucan,
It can be produced by a method including a step of oxidizing an α-glucan into which a group represented by the formula II has been introduced with periodic acid or a periodate to introduce an aldehyde group into the α-glucan. it can.
［ハイドロゲルの製造方法］
本発明のハイドロゲルは、上記製造された修飾α−グルカン化合物（ゲル化剤）、すなわち、式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入されてなる、ゲル化剤を、ポリチオール性還元剤によって架橋反応させて、ハイドロゲルを形成する工程、を含む方法によって、製造することができる。
[Manufacturing method of hydrogel]
The hydrogel of the present invention comprises the above-mentioned modified α-glucan compound (gelling agent) produced, that is, a gelling agent obtained by introducing a group represented by the formula II and an aldehyde group into α-glucan. It can be produced by a method including a step of forming a hydrogel by subjecting it to a cross-linking reaction with a sex reducing agent.
［ハイドロゲルの分解方法］
本発明のハイドロゲルは、優れた分解制御性を備えている。上記形成されたハイドロゲルに対して、ハイドロゲルの形成前又はハイドロゲルの形成後に、アミノ基を有する化合物を添加することによって、ハイドロゲルを制御して分解することができる。
[Hydrogel decomposition method]
The hydrogel of the present invention has excellent decomposition controllability. The hydrogel can be controlled and decomposed by adding a compound having an amino group to the formed hydrogel before the formation of the hydrogel or after the formation of the hydrogel.
［α−グルカン］
α−グルカンは、グルコースが脱水縮合してα結合により結合した糖鎖（多糖類）であり、例えば、デキストラン、デキストリン、プルランをあげることができる。好適な実施の態様において、重量平均分子量が、例えば、２０００〜２０万の範囲、８０００〜１５万の範囲、１万〜１０万の範囲にあるα−グルカンを使用できる。好適な実施の態様において、上記範囲の重量平均分子量であるデキストランを使用できる。重量平均分子量は、一般的な水系のＧＰＣ（ゲル濾過クロマトグラフィー）測定によって求めることができる。具体的には、実施例に開示の通りに、測定することができる。市販のデキストランとして、例えば、Pharmacosmos A/Sにより市販されている医療用グレードの製品、あるいは和光純薬により市販されている製品などを使用できる。
[Α-Glucan]
α-Glucan is a sugar chain (polysaccharide) in which glucose is dehydrated and condensed and bound by an α bond, and examples thereof include dextran, dextrin, and pullulan. In a preferred embodiment, α-glucans having a weight average molecular weight in the range of 20 to 200,000, 8000 to 150,000, and 10,000 to 100,000 can be used. In a preferred embodiment, dextran having a weight average molecular weight in the above range can be used. The weight average molecular weight can be determined by a general aqueous GPC (gel permeation chromatography) measurement. Specifically, it can be measured as disclosed in the examples. As the commercially available dextran, for example, a medical grade product marketed by Pharmacosmos A / S, a product marketed by Wako Pure Chemical Industries, etc. can be used.
［式Ｉの化合物］
式Ｉの化合物において、Ｒ１基は、例えばＣ１〜Ｃ３のアルキル基、Ｃ１〜Ｃ２のアルキル基とすることができ、例えばメチル基又はエチル基とすることができる。好適な実施の態様において、式Ｉの化合物は、グリシジルメタクリレート（ＧＭＡ）、又はグリシジルアクリレートとすることができる。
[Compound of formula I]
In the compound of formula I, the R1 group can be, for example, an alkyl group of C1 to C3, an alkyl group of C1 to C2, and can be, for example, a methyl group or an ethyl group. In a preferred embodiment, the compound of formula I can be glycidyl methacrylate (GMA) or glycidyl acrylate.
［式ＩＩの基の導入］
式Ｉの化合物によってα−グルカンへ導入される式ＩＩの基は、α−グルカンのグルコース単位のＯＨ基のなかのＨに置換されて、導入される。この導入率（ＤＳ％）は、1H-NMR測定によって、α−グルカンのグルコース単位あたりの式ＩＩの基の導入率（％）として、求めることができる。グルコース単位あたりの式ＩＩの基の導入率は、例えば、１０〜５０％の範囲、２０〜４０％の範囲とすることができる。なお、過ヨウ素酸酸化後の残存するグルコース単位あたりの導入率は、式ＩＩの基が導入されたグルコース単位と導入されていないグルコース単位とが同じ割合で過ヨウ素酸酸化による開裂を受けると換算すれば上記と同じ範囲となる。
[Introduction of group of formula II]
The group of formula II introduced into α-glucan by the compound of formula I is introduced by substituting H in the OH group of the glucose unit of α-glucan. This introduction rate (DS%) can be determined as the introduction rate (%) of the group of formula II per glucose unit of α-glucan by 1 H-NMR measurement. The introduction rate of the group of formula II per glucose unit can be, for example, in the range of 10 to 50% and in the range of 20 to 40%. The introduction rate per remaining glucose unit after periodic acid oxidation is calculated by converting the glucose unit into which the group of formula II is introduced and the glucose unit into which the group is not introduced to undergo cleavage due to periodic acid oxidation at the same ratio. If so, it will be in the same range as above.
α−グルカンと式Ｉの化合物との反応による式ＩＩの基の導入の反応は、例えばグリシジルメタクリレート（ＧＭＡ）と水酸基の一般的な反応条件で行うことができ、例えば窒素雰囲気中でジメチルスルホキシド（DMSO）とジメチルアミノピリジン(DMAP)の存在下で加熱して行うことができる。 The reaction of introducing a group of formula II by the reaction of α-glucan with a compound of formula I can be carried out under the general reaction conditions of, for example, glycidyl methacrylate (GMA) and a hydroxyl group, for example, dimethyl sulfoxide (in a nitrogen atmosphere). It can be carried out by heating in the presence of DMSO) and dimethylaminopyridine (DMAP).
［過ヨウ素酸酸化］
式ＩＩで表される基が導入されたα−グルカンを、過ヨウ素酸又は過ヨウ素酸塩で酸化して、アルデヒド基を、α−グルカンへ導入する。過ヨウ素酸酸化は、一般的な過ヨウ素酸酸化法の条件によって、実施できる。
[Periodic acid oxidation]
The α-glucan into which the group represented by the formula II has been introduced is oxidized with periodic acid or periodate to introduce an aldehyde group into the α-glucan. Periodic acid oxidation can be carried out under the conditions of a general periodic acid oxidation method.
［アルデヒド基の導入］
アルデヒド基は、α−グルカンのグルコース単位を過ヨウ素酸酸化によって開裂させて、導入される。この導入率（ＤＳ％）は、1H-NMR測定によって、α−グルカンのグルコース単位あたりのアルデヒド基の導入率（％）として、求めることができる。α−グルカンのグルコース単位あたりのアルデヒド基の導入率（％）は、例えば、２０〜８０％の範囲、２５〜７５％の範囲、４０〜６０％の範囲とすることができる。あるいは、過ヨウ素酸酸化を受けた後に残存するグルコース単位あたりのアルデヒド基の導入率として、この値を換算することもできる。この場合には残存するグルコース単位あたりのアルデヒド基の導入率として、例えば、２２（２２．２）〜１３３％の範囲、２９（２８．６）〜１２０％の範囲、５０〜８５．７（８６）％の範囲とすることができる。
[Introduction of aldehyde group]
The aldehyde group is introduced by cleaving the glucose unit of α-glucan by periodic acid oxidation. This introduction rate (DS%) can be determined as the introduction rate (%) of aldehyde groups per glucose unit of α-glucan by 1 H-NMR measurement. The introduction rate (%) of the aldehyde group per glucose unit of α-glucan can be, for example, in the range of 20 to 80%, in the range of 25 to 75%, and in the range of 40 to 60%. Alternatively, this value can be converted as the introduction rate of aldehyde groups per glucose unit remaining after undergoing periodic oxidation. In this case, the introduction rate of aldehyde groups per remaining glucose unit is, for example, in the range of 22 (22.2) to 133%, in the range of 29 (28.6) to 120%, and in the range of 50 to 85.7 (86). )% Can be in the range.
［架橋反応］
式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入された修飾α−グルカン化合物（ゲル化剤）は、ポリチオール性還元剤によって架橋反応させて、ハイドロゲルを形成できる。ポリチオール性還元剤としては、例えば、ポリチオール性アルコール、ポリチオールをあげることができ、例えば、ジチオール性アルコール、ジチオールをあげることができる。具体的には、例えば、ＤＴＴ（ジチオスレイトール）、１，４−ブタンジチオール、エタンチオール、１，１プロパンジチオールをあげることができる。ポリチオール性還元剤のＳＨ基は、式ＩＩの基と触媒なしにマイケル付加反応できる。これによって修飾α−グルカン化合物の分子が架橋されて、ハイドロゲルが形成される。この反応は不可逆反応であり、そのままでは分解は生じないために、このハイドロゲルは安定となる。
[Crossing reaction]
The modified α-glucan compound (gelling agent) in which the group represented by the formula II and the aldehyde group are introduced into α-glucan can be crosslinked with a polythiol reducing agent to form a hydrogel. Examples of the polythiol-based reducing agent include polythiol-based alcohol and polythiol, and examples thereof include dithiol-based alcohol and dithiol. Specifically, for example, DTT (dithiothreitol), 1,4-butanedithiol, ethanethiol, and 1,1propanedithiol can be mentioned. The SH group of the polythiol reducing agent can undergo a Michael addition reaction with the group of formula II without a catalyst. As a result, the molecules of the modified α-glucan compound are crosslinked to form a hydrogel. This reaction is an irreversible reaction, and decomposition does not occur as it is, so that the hydrogel is stable.
［ハイドロゲルの安定性］
本発明のハイドロゲルは、架橋反応によって形成された架橋がそのままでは分解しないという点で安定である。例えば、ＰＢＳ（リン酸緩衝生理食塩水）に３７℃で８日間保温しても約８０パーセントのゲルが維持されている。
[Hydrogel stability]
The hydrogel of the present invention is stable in that the crosslinks formed by the crosslink reaction do not decompose as they are. For example, about 80 percent of the gel is maintained even when heated in PBS (phosphate buffered saline) at 37 ° C. for 8 days.
［ハイドロゲルの分解性］
本発明のハイドロゲルには、導入されたアルデヒド基が、架橋反応に使用されることなく、保持されている。そして、このアルデヒド基に対して、アミノ基を反応させると、α−グルカン構造の主鎖が切断されて、分子量の低下を伴う断片化が生じて、結果として、ハイドロゲルが分解される。
[Degradability of hydrogel]
In the hydrogel of the present invention, the introduced aldehyde group is retained without being used in the cross-linking reaction. Then, when an amino group is reacted with this aldehyde group, the main chain of the α-glucan structure is cleaved, fragmentation accompanied by a decrease in molecular weight occurs, and as a result, the hydrogel is decomposed.
［ハイドロゲルの分解制御性］
本発明のハイドロゲルは、導入されたアルデヒド基に対して、アミノ基を反応させることによって、その分解を制御することができる。アミノ基の添加には、例えば、ハイドロゲルの形成前に、又はハイドロゲルの形成後に、アミノ基を有する化合物を添加することによって行うことができる。例えば、実施例に示す通り、アミノ基を有する化合物としてグリシンを添加した場合には、その濃度に依存して、３７℃で８日間保温後のハイドロゲルの残存を、約６０％から０％という広範囲にわたって制御している。
[Hydrogel decomposition controllability]
The hydrogel of the present invention can control its decomposition by reacting the introduced aldehyde group with an amino group. The addition of the amino group can be carried out, for example, by adding a compound having an amino group before the formation of the hydrogel or after the formation of the hydrogel. For example, as shown in Examples, when glycine is added as a compound having an amino group, the residual hydrogel after heat retention at 37 ° C. for 8 days is said to be about 60% to 0%, depending on the concentration thereof. It is controlled over a wide range.
［アミノ基を有する化合物］
ハイドロゲルの分解制御のために添加されるアミノ基を有する化合物としては、特段に制限はない。生体内にハイドロゲルを埋め込む場合には、生体内において接触するアミノ基を考慮して、分解制御することができる。アミノ基を有する化合物としては、アミノ酸やタンパク質などのアミノ基を有する生体分子であってもよく、アミノ基を付加した生体分子であってもよく、アミノ基を付加した人工の分子であってもよい。好適な実施の態様において、アミノ酸、及びアミノ基を付加したアミノ化多糖類をあげることができる。アミノ酸として、グリシンなどの天然のアミノ酸、非天然のアミノ酸をあげることができる。アミノ基を付加したアミノ化多糖類として、例えばアミノ化κカラギーナンをあげることができる。
[Compound having an amino group]
The compound having an amino group added for controlling the decomposition of the hydrogel is not particularly limited. When implanting a hydrogel in a living body, decomposition control can be performed in consideration of amino groups that come into contact with the living body. The compound having an amino group may be a biomolecule having an amino group such as an amino acid or a protein, a biomolecule having an amino group added, or an artificial molecule having an amino group added. Good. In a preferred embodiment, amino acids and amination polysaccharides to which an amino group has been added can be mentioned. Examples of amino acids include natural amino acids such as glycine and unnatural amino acids. Examples of the amination polysaccharide to which an amino group is added include aminated reductive carrageenan.
［アミノ化κカラギーナン］
カラギーナンは、ゲルを形成することが知られている。アミノ化κカラギーナンは、３７℃ではゲル状態を維持して、数度の温度の昇温で溶解するゲルを作成することができる。そこで、アミノ化κカラギーナンによる微小なゲルをあらかじめ調製して、これを本発明のハイドロゲル中に分散させておき、所望により数度程度の加熱の操作を行うことで、アミノ化κカラギーナンのゲルが溶解して、アミノ化κカラギーナンのアミノ基が露出して、本発明のハイドロゲルのアルデヒド基と自由に反応できるアミノ基が増大することによって、本発明のハイドロゲルの速やかな分解を可能とする。この態様によれば、ハイドロゲルにアミノ基を有する化合物を添加していても、その後の任意の時点から、ゲルの分解の反応を開始又は加速することができる。すなわち、アミノ基を有する化合物として、アミノ化κカラギーナンを使用した態様もまた、本発明の範囲内にある。
[Amination κ carrageenan]
Carrageenan is known to form gels. Aminated reductive carrageenan can maintain a gel state at 37 ° C. to prepare a gel that dissolves at a temperature rise of several degrees Celsius. Therefore, a fine gel made of aminated κ carrageenan is prepared in advance, dispersed in the hydrogel of the present invention, and if desired, a heating operation of several degrees is performed to gel the aminated κ carrageenan. Dissolves and exposes the amino group of aminated κ carrageenan, increasing the number of amino groups that can freely react with the aldehyde group of the hydrogel of the present invention, thereby enabling rapid decomposition of the hydrogel of the present invention. To do. According to this aspect, even if a compound having an amino group is added to the hydrogel, the reaction of gel decomposition can be started or accelerated from any subsequent time point. That is, an embodiment using aminated kappa carrageenan as a compound having an amino group is also within the scope of the present invention.
［分解制御性ハイドロゲル］
本発明の分解制御性ハイドロゲルは、α−グルカンに基づいているために生体親和性が高く、優れた分解制御性を備えるために、医療用接着剤、ＤＤＳ基材として好適に使用できる。したがって、本発明は、分解制御性ハイドロゲルからなる、医療用接着剤にもあり、ＤＤＳ基材にもある。
[Decomposition controllable hydrogel]
Since the decomposition controllable hydrogel of the present invention is based on α-glucan, it has high biocompatibility and has excellent decomposition controllability, so that it can be suitably used as a medical adhesive and a DDS base material. Therefore, the present invention is also found in medical adhesives made of degradation-controllable hydrogels and in DDS substrates.
以下に実施例をあげて、本発明を詳細に説明する。本発明は、以下に例示する実施例に限定されるものではない。なお、実施例中、特にことわりのない限り「％」及び「部」はそれぞれ重量％及び重量部を示す。 Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below. In the examples, “%” and “parts” indicate% by weight and parts by weight, respectively, unless otherwise specified.
［実験例１］
［メタクリル酸グリシジル導入デキストラン(Dex-GMA)の合成］
5gのデキストラン（名糖産業、分子量70000）を20mLのジメチルスルホキシド（DMSO）に溶解し、窒素ガスを３０分吹き込んだ。4.8gのジメチルアミノピリジン(DMAP)と2.3gのグリシジルメタクリレート（GMA）を添加し、３０分チッソ雰囲気下で反応させた。溶液を50℃に熱し、１２時間さらに反応させた後、HClを添加してDMAPを中和し反応を停止させた。溶液はMWCO=3500の透析膜（Spectra/Por）で蒸留水に対して一週間透析を行い、続けて凍結乾燥により反応したデキストラン(Dex-GMA)を精製回収した。得られたDex-GMAに対して、FTIRと1H-NMRでキャラクタリゼーションを行った。
[Experimental Example 1]
[Synthesis of glycidyl methacrylate-introduced dextran (Dex-GMA)]
5 g of dextran (Meito Sangyo, molecular weight 70000) was dissolved in 20 mL of dimethyl sulfoxide (DMSO), and nitrogen gas was blown in for 30 minutes. 4.8 g of dimethylaminopyridine (DMAP) and 2.3 g of glycidyl methacrylate (GMA) were added and reacted in a nitrogen atmosphere for 30 minutes. The solution was heated to 50 ° C. and further reacted for 12 hours, after which HCl was added to neutralize DMAP and terminate the reaction. The solution was dialyzed against distilled water for one week with a dialysis membrane (Spectra / Por) of MWCO = 3500, and then dextran (Dex-GMA) reacted by freeze-drying was purified and recovered. The obtained Dex-GMA was characterized by FTIR and 1 H-NMR.
FTIRチャートにおいて、1707cm-1付近に新たに出現したC=OバンドからGMAの導入を定性的に確認し、導入率（Degree of substitution, DS）は１H-NMRスペクトルで、アノメリックプロトンのピーク（5.08ppm）に対するビニルプロトンのピーク（6.32-6.34ppm）の割合から算出した。その結果は、29%（グルコースユニット当たり）であった。 In the FTIR chart, the introduction of GMA was qualitatively confirmed from the newly appearing C = O band near 1707 cm -1 , and the introduction rate (Degree of substitution, DS) was 1H-NMR spectrum, and the peak of anomeric proton ( It was calculated from the ratio of the peak of vinyl protons (6.32-6.34ppm) to 5.08ppm). The result was 29% (per glucose unit).
上記のDex-GMA合成の反応を、図１に示す。上記の1H-NMR測定の結果を、図２に示す。上記のFTIR測定の結果を、図３に示す。上記GMAの導入率（DS）は図３に示す１H-NMRスペクトルのピークから、ＤＳ（％）＝（［Ｈｂ］／［Ｈａ］）×１００（％）の式によって算出した。 The reaction of the above Dex-GMA synthesis is shown in FIG. The results of the above 1 H-NMR measurement are shown in FIG. The result of the above FTIR measurement is shown in FIG. The introduction rate (DS) of GMA was calculated from the peak of the 1H-NMR spectrum shown in FIG. 3 by the formula of DS (%) = ([Hb] / [Ha]) × 100 (%).
［実験例２］
［Dex-GMAとジチオトレイトール(DTT)との反応によるゲル化］
10wt%のDex-GMA水溶液と1.36wt%のDTT水溶液をそれぞれ0.1mLずつ混合することで、2時間55分でゲル化が起こった。ゲル化は、混合溶液を入れた試験管を反転して溶液が落下しなくなるまでの時間とした。また、10wtのDex-GMA0.2mLと1.36%のDTT0.1mLを混合すると2時間8分でゲル化することを確認した。
[Experimental Example 2]
[Gelification by reaction between Dex-GMA and dithiothreitol (DTT)]
By mixing 0.1 mL each of a 10 wt% Dex-GMA aqueous solution and a 1.36 wt% DTT aqueous solution, gelation occurred in 2 hours and 55 minutes. Gelation was performed by inverting the test tube containing the mixed solution until the solution did not fall. It was also confirmed that when 0.2 mL of 10 wt Dex-GMA and 0.1 mL of 1.36% DTT were mixed, gelation occurred in 2 hours and 8 minutes.
上記のDex-GMAのゲル化の反応を図４に示す。上記のゲル化の実験結果をまとめて、図５に示す。 The gelation reaction of Dex-GMA described above is shown in FIG. The results of the above gelation experiment are summarized in FIG.
［実験例３］
［Ox-Dex-GMAの合成］
酸化Dex-GMA（Ox-Dex-GMA）の合成は以下の手法で行った。
実験例１で作成したDex-GMA2.5gを20mLの蒸留水に溶解し、0.75gの過ヨウ素酸ナトリウムを溶解した20mLの蒸留水と混合し、50度で1時間反応させた。反応後はMWCO3500の透析膜で蒸留水に対し1日間透析を行い、凍結乾燥でOx-Dex-GMAを回収した。キャラクタリゼーションは１H-NMRで行った。１H-NMRで9.64ppmのアルデヒドピークでアルデヒドの導入を確認した。アルデヒド基の導入量の定量化は、アンモニア存在化でのアセトアセトアニリド（AAA）とアルデヒドの反応による蛍光の検出により算出した（Li et al., Analytical Sciences, 2007, 23, 1810-1860）ところ、DS（導入率）は51%であった。
[Experimental Example 3]
[Synthesis of Ox-Dex-GMA]
Oxidized Dex-GMA (Ox-Dex-GMA) was synthesized by the following method.
2.5 g of Dex-GMA prepared in Experimental Example 1 was dissolved in 20 mL of distilled water, mixed with 20 mL of distilled water in which 0.75 g of sodium periodate was dissolved, and reacted at 50 ° C. for 1 hour. After the reaction, distilled water was dialyzed against distilled water with a dialysis membrane of MWCO 3500 for 1 day, and Ox-Dex-GMA was recovered by freeze-drying. The characterization was performed by 1H-NMR. Aldehyde introduction was confirmed by 1H-NMR with an aldehyde peak of 9.64 ppm. The quantification of the amount of aldehyde group introduced was calculated by detecting fluorescence due to the reaction of acetanilide (AAA) and aldehyde in the presence of ammonia (Li et al., Analytical Sciences, 2007, 23, 1810-1860). The DS (introduction rate) was 51%.
上記のOx-Dex-GMA合成の反応を、図６に示す。上記の1H-NMR測定の結果と、上記のFTIR測定の結果をまとめて、図７に示す。上記アルデヒド基の導入率（ＤＳ）は、図７に示す１H-NMRにおいて、ＤＳ（％）＝（［Ｈｄ］／［Ｈａ］）×１００（％）の式によって求めた。 The reaction of the above Ox-Dex-GMA synthesis is shown in FIG. The results of the above 1 H-NMR measurement and the results of the above FTIR measurement are summarized in FIG. 7. The introduction rate (DS) of the aldehyde group was determined by the formula DS (%) = ([Hd] / [Ha]) × 100 (%) in 1H-NMR shown in FIG.
［比較実験例１］
［Ox-Dexの合成］
Dex2.5gを20mLの蒸留水に溶解し、0.75gの過ヨウ素酸ナトリウムを溶解した20mLの蒸留水と混合し、50度で1時間反応させた。反応後はMWCO3500の透析膜で蒸留水に対し1日間透析を行い、凍結乾燥でOx-Dexを回収した。キャラクタリゼーションは実験例３に準じた方法で行い、DS（導入率）は52%であった。
[Comparative Experiment Example 1]
[Synthesis of Ox-Dex]
2.5 g of Dex was dissolved in 20 mL of distilled water, 0.75 g of sodium periodate was mixed with 20 mL of distilled water, and the mixture was reacted at 50 ° C. for 1 hour. After the reaction, distilled water was dialyzed against distilled water with a dialysis membrane of MWCO 3500 for 1 day, and Ox-Dex was recovered by freeze-drying. The characterization was performed by the method according to Experimental Example 3, and the DS (introduction rate) was 52%.
［実験例４］
［Ox-Dex-GMAとDTTの反応によるゲル化］
10wt%のOx-Dex-GMA水溶液と1.36wt%のDTT水溶液を0.1mLずつ混合することで、37度において45分でゲル化を確認した。また、Ox-Dex-GMAを0.2mLとしたときのゲル化時間は10時間40分、一方DTTを0.2mLとしたときのゲル化時間は20分であった。これらのゲルはPBS中で分解せず安定であった。
[Experimental Example 4]
[Gelification by reaction between Ox-Dex-GMA and DTT]
By mixing 0.1 mL each of a 10 wt% Ox-Dex-GMA aqueous solution and a 1.36 wt% DTT aqueous solution, gelation was confirmed at 37 ° C. in 45 minutes. The gelation time when Ox-Dex-GMA was 0.2 mL was 10 hours and 40 minutes, while the gelation time when DTT was 0.2 mL was 20 minutes. These gels did not decompose in PBS and were stable.
上記のOx-Dex-GMAのゲル化の反応を図８に示す。上記のゲル化の実験結果をまとめて、図９に示す。図９に示されるように、Ox-Dex-GMAと架橋剤の濃度を変化させることによって、ゲル化時間を制御することができた。 The gelation reaction of Ox-Dex-GMA described above is shown in FIG. The results of the above gelation experiment are summarized in FIG. As shown in FIG. 9, the gelation time could be controlled by changing the concentration of Ox-Dex-GMA and the cross-linking agent.
［実験例５］
［アミノ化κカラギーナンの合成］
アミノ化κカラギーナン（amino-CG）はカラギーナン（CG）から以下のように合成した。κ（カッパ）カラギーナン（東京化成）1gを100mLのナス型フラスコ中10mLの2-プロパノールに分散させ、40度で40%NaOH溶液を1.2mLゆっくりと滴下したのち1時間リフラックスさせ反応させた。その後、3-ブロモプロピルアミンを0.547g添加し、50度で24時間反応させた。反応終了後、１Mの塩酸で中和し、得られた沈殿物をフィルターで集め、2-プロパノールで洗浄し、凍結乾燥によりアミノ化カラギーナンを回収した。キャラクタリゼーションは１H-NMRとFTIRにより行った。１H-NMRでは1.5-2.25ppm付近にアミノ基に隣接したメチレンプロトンによるピークが確認できた。FTIRスペクトルでは、1563cm-1付近に一級アミンによる新しいバンドが確認できた。DSはアミノ基の定量法であるTNBS法（Means GR et al., Amino groups. In Chemical Modification of Proteins, Holden-Day, Inc.: San Francisco, 1971, pp214-217）により求めたところ、0.87%であった。
[Experimental Example 5]
[Synthesis of aminated reductive carrageenan]
Aminated reductive carrageenan (amino-CG) was synthesized from carrageenan (CG) as follows. 1 g of κ (kappa) carrageenan (Tokyo Kasei) was dispersed in 10 mL of 2-propanol in a 100 mL eggplant-shaped flask, 1.2 mL of 40% NaOH solution was slowly added dropwise at 40 ° C, and then refluxed for 1 hour for reaction. Then, 0.547 g of 3-bromopropylamine was added, and the mixture was reacted at 50 ° C. for 24 hours. After completion of the reaction, the reaction was neutralized with 1 M hydrochloric acid, the obtained precipitate was collected with a filter, washed with 2-propanol, and aminated carrageenan was recovered by lyophilization. The characterization was performed by 1H-NMR and FTIR. In 1H-NMR, a peak due to methylene proton adjacent to the amino group was confirmed at around 1.5-2.25 ppm. In the FTIR spectrum, a new band due to the primary amine was confirmed around 1563 cm -1 . DS was determined by the TNBS method (Means GR et al., Amino groups. In Chemical Modification of Proteins, Holden-Day, Inc .: San Francisco, 1971, pp214-217), which is a method for quantifying amino groups, and found that it was 0.87%. Met.
上記のアミノ化κカラギーナンの合成の反応を、図１０に示す。上記の1H-NMR測定の結果を、図１１に示す。上記のFTIR測定の結果を、図１２に示す。上記のＴＮＢＳ法による測定の原理を、図１３に示す。 The reaction for the synthesis of the above aminated reductive carrageenan is shown in FIG. The result of the above 1 H-NMR measurement is shown in FIG. The result of the above FTIR measurement is shown in FIG. The principle of measurement by the above TNBS method is shown in FIG.
［実験例６］
［ゲルの分解挙動］
0.5mLの10wt%Ox-Dex-GMA水溶液と0.5mLの1.36wt%のDTT水溶液を混合し、ゲル化させた。得られたゲルに、PBS、1-10%のグリシン水溶液、又は1%のアミノ化カラギーナン水溶液の何れかを3mL添加し、時間ごとにゲルを取り出し、乾燥重量を測定することで元のゲルの乾燥重量との比からゲルの残存重量を計算した。Ox-Dex-GMAのゲルについて、PBSを添加した場合にはアルデヒドが残存しているため、分解反応が起こらず、ゲルの残存重量は8日後に80%であった。一方、Ox-Dex-GMAのゲルについて、グリシンを添加した場合、1%グリシンだと8日後に60.7%まで重量が減少し、5％グリシンでは6日後にゲルがすべて溶解した。10%グリシン添加系では4日後に消失した。1%アミノ化カラギーナン添加系では8日後に51.1％とゲルの分解が確認されている。ゲル化反応では使用されなかったアルデヒド基が、グリシンもしくはアミノ化カラギーナンのアミノ基と反応することで分子鎖の分解がスタートした結果であると考えられる。
同様の実験を、10wt%Ox-Dex-GMA水溶液に代えて、0.5mLの10wt%Ox-Dex水溶液を使用して、同様に0.5mLの1.36wt%のDTT水溶液を混合し、ゲル化させて行った。Ox-Dex-GMAのゲルについては、PBSを添加した。Ox-Dexゲル場合、PBS中で6日後にはゲルの消失が見られている。これは、DTTとOx-Dexのゲルではアルデヒド基がゲル化反応に使用されているため、分子鎖の分解が起こっていることが考えられる。
[Experimental Example 6]
[Gel decomposition behavior]
A 0.5 mL 10 wt% Ox-Dex-GMA aqueous solution and a 0.5 mL 1.36 wt% DTT aqueous solution were mixed and gelled. To the obtained gel, add 3 mL of either PBS, 1-10% glycine aqueous solution, or 1% aminated carrageenan aqueous solution, take out the gel every hour, and measure the dry weight to obtain the original gel. The residual weight of the gel was calculated from the ratio to the dry weight. Regarding the Ox-Dex-GMA gel, when PBS was added, aldehyde remained, so no decomposition reaction occurred, and the residual weight of the gel was 80% after 8 days. On the other hand, when glycine was added to the Ox-Dex-GMA gel, the weight of 1% glycine decreased to 60.7% after 8 days, and that of 5% glycine completely dissolved the gel after 6 days. It disappeared after 4 days in the 10% glycine-added system. In the 1% amination carrageenan addition system, gel decomposition was confirmed to be 51.1% after 8 days. It is considered that the aldehyde group, which was not used in the gelation reaction, reacted with the amino group of glycine or carrageenan amination to start the decomposition of the molecular chain.
In a similar experiment, a 0.5 mL 10 wt% Ox-Dex aqueous solution was used instead of the 10 wt% Ox-Dex-GMA aqueous solution, and a 0.5 mL 1.36 wt% DTT aqueous solution was also mixed and gelled. went. For Ox-Dex-GMA gels, PBS was added. In the case of Ox-Dex gel, disappearance of the gel was observed in PBS after 6 days. This is because the aldehyde group is used in the gelation reaction in the gel of DTT and Ox-Dex, so it is considered that the molecular chain is decomposed.
上記のゲル化と分解の実験の操作の流れを、図１４と図１６に示す。上記のゲルの分解率と経過時間の結果を、図１５と図１７に示す。Ox-Dex-GMA を用いたハイドロゲルは、ＰＢＳ中で安定であったが、アミノ基の添加によって分解可能であった。 The operation flow of the above gelation and decomposition experiment is shown in FIGS. 14 and 16. The results of the decomposition rate and elapsed time of the above gel are shown in FIGS. 15 and 17. Hydrogels with Ox-Dex-GMA were stable in PBS but degradable with the addition of amino groups.
［実験例７］
［分子鎖の分解の確認］
2wt%のOx-Dex-GMA水溶液に対して、5wt%のグリシン水溶液、5wt%のアミノ化カラギーナン水溶液、又は5wt%のアセチルシステイン水溶液の何れかを等量混合し、ゲルパーミエーションクロマトグラフィー（GPC、島津）にて120分間の分子量の推移を20分ごとに測定した。移動相液体はPBS、流速は1mL/minとし、カラムはBioSep-2000（Phenomenex）を使用した。その結果、グリシンを添加したOx-Dex-GMAと、アミノ化カラギーナンを添加したOx-Dex-GMAは、分子量が低下したのに対し、アセチルシステインを添加した系では分子量の低下は見られなかった。
[Experimental Example 7]
[Confirmation of molecular chain decomposition]
Gel permeation chromatography (GPC) is performed by mixing an equal amount of 5 wt% glycine aqueous solution, 5 wt% amino carrageenan aqueous solution, or 5 wt% acetylcysteine aqueous solution with 2 wt% Ox-Dex-GMA aqueous solution. , Shimadzu), the transition of molecular weight for 120 minutes was measured every 20 minutes. The mobile phase liquid was PBS, the flow rate was 1 mL / min, and the column was BioSep-2000 (Phenomenex). As a result, the molecular weights of Ox-Dex-GMA to which glycine was added and Ox-Dex-GMA to which amination carrageenan was added decreased, whereas the molecular weight of the system to which acetylcysteine was added did not decrease. ..
これは、アミノ基がアルデヒドと反応し、その反応が主鎖分解の開始反応であることを支持する結果であった。一方でアセチルシステインの場合、SHはGMA部位と反応することでアルデヒドは反応に寄与せず、分解反応が起こらなかったと考えられる。以上のことから、ゲルにおいてもDTTのSHがGMAと優先的に反応し、アルデヒドがゲル化に関与せずに残存しており、後からグリシンやアミノ化カラギーナンを加えることで分解の開始反応を制御できることが分かった。 This was a result supporting that the amino group reacted with the aldehyde and the reaction was the initiation reaction of the main chain decomposition. On the other hand, in the case of acetylcysteine, it is considered that SH did not contribute to the reaction because SH reacted with the GMA site, and the decomposition reaction did not occur. From the above, SH of DTT reacts preferentially with GMA in the gel, and aldehyde remains without being involved in gelation. Later, by adding glycine or carrageenan amination, the decomposition initiation reaction is started. It turned out to be controllable.
アセチルシステインの構造式を後述して示す。上記のGPCによる測定の結果を、図１８に示す。Ox-Dex-GMAにSH基を添加しても分子量（Mw）は低下しないが、アミノ基（グリシンもしくはアミノカラギーナンに含まれるアミノ基）の添加で分子量の低下（分解）が見られた。 The structural formula of acetylcysteine will be shown later. The result of the above measurement by GPC is shown in FIG. The molecular weight (Mw) did not decrease when the SH group was added to Ox-Dex-GMA, but the molecular weight decreased (decomposition) when the amino group (amino group contained in glycine or aminocarrageenan) was added.
アセチルシステイン
本発明は、分解制御性を備えたハイドロゲルを提供する。本発明は産業上有用な発明である。 The present invention provides a hydrogel having decomposition controllability. The present invention is an industrially useful invention.
Claims (8)
（ただし、式Ｉ中、Ｒ１基は、Ｃ１〜Ｃ３のアルキル基である）
で表される化合物を反応させて、次の式ＩＩ：
（ただし、式ＩＩ中、Ｒ１基は、式Ｉ中のＲ１基と同一の基である）
で表される基を、α−グルカンへ導入する工程、
式ＩＩで表される基が導入されたα−グルカンを、過ヨウ素酸又は過ヨウ素酸塩で酸化して、アルデヒド基を、α−グルカンへ導入する工程、
を含む、式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入されてなる、ゲル化剤の製造方法によって製造された、式ＩＩで表される基及びアルデヒド基がα−グルカンへ導入されてなる、ゲル化剤を、ポリチオール性還元剤によって架橋反応させて、ハイドロゲルを形成する工程、
を含む、ハイドロゲルの製造方法。 Α-Glucan having a weight average molecular weight in the range of 2000 to 200,000 and the following formula I:
(However, in Formula I, the R1 group is an alkyl group of C1 to C3)
By reacting the compound represented by, the following formula II:
(However, in Formula II, the R1 group is the same group as the R1 group in Formula I)
The process of introducing the group represented by (1) into α-glucan,
A step of oxidizing an α-glucan into which a group represented by the formula II has been introduced with periodic acid or a periodate to introduce an aldehyde group into the α-glucan.
The group represented by the formula II and the aldehyde group are introduced into the α-glucan, and the group represented by the formula II and the aldehyde group produced by the method for producing a gelling agent are introduced into the α-glucan. The step of forming a hydrogel by cross-linking the gelling agent with a polythiol reducing agent.
A method for producing a hydrogel, including .
（ただし、式ＩＩ中、Ｒ１基は、Ｃ１〜Ｃ３のアルキル基である）
で表される基が、α−グルカン中のＯＨ基のＨに置換されて、α−グルカンのグルコース単位あたり１０〜５０％の範囲の導入率で導入され、
過ヨウ素酸酸化によるアルデヒド基が、α−グルカンのグルコース単位あたり２５〜７５％の範囲の導入率で導入されてなる、修飾α−グルカン化合物が、ジチオスレイトールによって架橋されてなる、ハイドロゲル。 For α-glucans with a weight average molecular weight in the range of 2000-200,000, the following formula II:
(However, in Formula II, the R1 group is an alkyl group of C1 to C3).
The group represented by is substituted with H of the OH group in α-glucan and introduced at an introduction rate in the range of 10 to 50% per glucose unit of α-glucan.
A hydrogel in which a modified α-glucan compound is crosslinked by dithioslateol, in which an aldehyde group due to periodic oxidation is introduced at an introduction rate in the range of 25-75% per glucose unit of α-glucan .
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