TW202227538A - 具有胺基之聚輪烷 - Google Patents
具有胺基之聚輪烷 Download PDFInfo
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- TW202227538A TW202227538A TW110143701A TW110143701A TW202227538A TW 202227538 A TW202227538 A TW 202227538A TW 110143701 A TW110143701 A TW 110143701A TW 110143701 A TW110143701 A TW 110143701A TW 202227538 A TW202227538 A TW 202227538A
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Abstract
Description
本發明關於具有胺基之聚輪烷。
細胞培養技術在醫藥品及化粧品領域、再生醫療研究中被廣泛利用,為重要的技術。就細胞培養之環境而言,需要適當的培養環境、培養液、培養基材。其中,培養基材具有作為細胞黏附的支架之作用,其是否為適於細胞培養之表面?能否加工?有無細胞毒性?能否滅菌處理?並要求可維持滅菌狀態、於培養條件下不會變質、不會妨礙顯微鏡的觀察等特性。
以往,就培養基材而言,玻璃產品被大量使用,但目前為聚苯乙烯產品被廣泛使用。但是,將聚苯乙烯樹脂予以成形而得的培養基材,其表面為疏水性,和細胞之親和性亦低,故一般而言會施加表面處理。表面處理可列舉:電漿處理、電暈放電處理、氧化劑處理、親水性物質所為之塗佈。塗佈劑多為促進細胞黏附者,例如已知有I型膠原蛋白、IV型膠原蛋白、明膠、纖連蛋白(fibronectin)、玻連蛋白(vitronectin)、層黏連蛋白、基質膠(matrigel)、羥基磷灰石等。又,亦已知以水溶性彈性蛋白進行塗佈的話,會促進血管平滑肌細胞或彈性蛋白反應性細胞之分化誘導。
間葉系幹細胞係具有自我複製能力及分化成成骨細胞、軟骨細胞、脂肪細胞、骨骼肌細胞、韌帶細胞等間葉系組織之分化多能性之來自中胚層之成體幹細胞。在成體組織,係存在於真皮、骨骼肌、脂肪組織等結締組織中、或主要存在於骨髓間質中,並對結締組織之修復、恆定性的維持、造血幹細胞之增殖或分化的控制發揮功能。間葉系幹細胞的分化係由生物學性因子、物理學性因子複雜地進行控制。例如已知在間葉系幹細胞之培養基中添加地塞米松(dexamethasone)、β-甘油磷酸、抗壞血酸的話,可分化誘導成成骨細胞;若添加地塞米松、3-異丁基-1-甲基黃嘌呤、胰島素、吲哚美辛(indomethacin)的話,可分化誘導成脂肪細胞。
近年,培養基材的特性(例如硬度等整體特性、塗佈之表面特性)會對培養細胞的功能造成影響正受到關注,且已報告使具有期望的功能之細胞增殖之研究(非專利文獻1~3)。
[先前技術文獻]
[非專利文獻]
[非專利文獻1]Cell, 2006, 126, 677-689.
[非專利文獻2]Nature Reviews Materials, 2020, 5, 686-705.
[非專利文獻3]Adv. Healthcare Mater. 2015, 4, 215-222.
[發明所欲解決之課題]
但是,為了獲得具有期望的功能之細胞而微調細胞培養基材的整體特性(硬度等),迄今仍為困難。據認為為了使間葉系幹細胞分化成期望的細胞系統,選擇適當的物理學性因子係為有效。於是,本發明的目的係提供可促進間葉系幹細胞之分化誘導的塗佈劑及培養基材。
[解決課題之手段]
本發明提供如下之[1]~[15]。
[1] 一種聚輪烷,係以式(1)表示。
[化1]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化2]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。
[2] 一種塗佈劑,含有式(1)表示之聚輪烷。
[化3]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化4]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。
[3] 如[2]所記載之塗佈劑,其中,前述聚輪烷含有1~18個羥基被-X-NH
2表示之基修飾而成的環糊精。
[4] 如[2]或[3]所記載之塗佈劑,其中,前述聚輪烷中,環糊精的貫穿數為3~220個。
[5] 一種細胞培養方法,用以促進間葉系幹細胞之脂肪分化;
包含在經含有式(1)表示之聚輪烷的組成物塗佈後之基板的表面上培養間葉系幹細胞之步驟。
[化5]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化6]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。
[6] 如[5]所記載之細胞培養方法,其包含在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
[7] 如[5]或[6]所記載之細胞培養方法,其中,前述聚輪烷含有1~18個羥基被-X-NH
2表示之基修飾而成的環糊精。
[8] 如[5]~[7]中任一項所記載之細胞培養方法,其中,前述聚輪烷中,環糊精的貫穿數為3~220個。
[9] 一種細胞培養方法,用以抑制間葉系幹細胞之成骨分化;
包含在經含有式(1)表示之聚輪烷的組成物塗佈後之基板的表面上培養間葉系幹細胞之步驟。
[化7]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化8]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。
[10] 如[9]所記載之細胞培養方法,其包含在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
[11] 如[9]或[10]所記載之細胞培養方法,其中,前述聚輪烷含有1~18個羥基被-X-NH
2表示之基修飾而成的環糊精。
[12] 如[9]~[11]中任一項所記載之細胞培養方法,其中,前述聚輪烷中,環糊精的貫穿數為3~220個。
[13] 一種培養基材,包含經含有式(1)表示之聚輪烷的組成物塗佈後之基板。
[化9]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化10]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。
[14] 如[13]所記載之培養基材,其中,前述聚輪烷含有1~18個羥基被-X-NH
2表示之基修飾而成的環糊精。
[15] 如[13]或[14]所記載之培養基材,其中,前述聚輪烷中,環糊精的貫穿數為3~200個。
[發明之效果]
根據本發明,可提供能抑制間葉系幹細胞之成骨分化或能促進脂肪分化之塗佈劑。又,根據本發明,還可提供經該塗佈劑塗佈後之基板(培養基材)。
<第一實施形態>
本發明之第一實施形態係式(1)表示之聚輪烷。
[化11]
式中,R
1為氫原子或甲基,m為1~2000,n為10~500,
[化12]
為至少1個羥基被-X-NH
2表示之基修飾而成的環糊精,X為2價有機基。聚輪烷1分子中含有至少1個該環糊精。
式(1)表示之聚輪烷係由軸分子(線狀高分子)及經修飾之環糊精(CD,環狀分子)構成,軸分子貫穿至少1個經修飾之環糊精,並於其兩端予以封端。
軸分子係以下式(2)表示,具有於分子中央具有聚乙二醇結構且末端被苯基二硫代酯基封端之聚(甲基)丙烯酸酯結構。式中,R
1為氫原子或甲基。環糊精係在聚乙二醇結構部分被貫穿。各R
1可互為相同也可相異。R
1宜為甲基。
[化13]
聚乙二醇結構若含有乙二醇作為單體單元且具備可貫穿至少1個環糊精之分子長即可。形成聚乙二醇結構之乙二醇單元的數量可為20~1000,宜為85~800,為100~700更佳。式中,n可為10~500,宜為43~400,為50~350更佳。
聚(甲基)丙烯酸酯結構包含(甲基)丙烯酸苄酯作為單體單元。本說明書中,「(甲基)丙烯酸酯」一詞意指「丙烯酸酯」及「甲基丙烯酸酯」之兩者。發明人們認為聚(甲基)丙烯酸酯結構具有提高對聚苯乙烯等基板之密接性的作用,故貫穿環糊精的聚乙二醇部分成為以環套狀地從基板分離的狀態。形成聚(甲基)丙烯酸酯結構之(甲基)丙烯酸苄酯單元的數目m,就每個三嵌段共聚物之一末端的聚(甲基)丙烯酸苄酯而言,可為1~2000,宜為20~1000,為30~500更佳。各m可互為相同也可相異。
本實施形態所使用的經修飾之環糊精係以下式(3)表示,構成成分之葡萄糖的羥基之至少1個被-X-NH
2修飾。環糊精為α-環糊精、β-環糊精、γ-環糊精及它們的組合中任一者皆可。理想的環糊精為α-環糊精。下述為示意性地表示經-X-NH
2修飾之環糊精的圖。圖中的「-O-X-NH
2」表示構成環糊精之葡萄糖中的羥基被-X-NH
2修飾。圖中,「-O-X-NH
2」僅顯示1個,但不應被限定性地解釋成只有1個羥基被-X-NH
2修飾。
[化14]
在聚輪烷中,至少1個經修飾之環糊精被軸分子貫穿。每1分子聚輪烷的環糊精之數量可獨立地定義,係取決於聚乙二醇結構的分子長,而不必唯一地定義。又,就化學計量而言,環糊精能包接聚乙二醇之重複單元即乙二醇之2單元,故環狀分子的數量取決於所使用的線狀高分子之分子量而有其上限。例如,就每1分子數目平均分子量20000的聚乙二醇而言之環糊精的數量可為3~220,宜為5~150,為5~120更佳,為5~100特佳。惟,環糊精的軸分子之主鏈方向的長度(聚輪烷具有多個環糊精時,為其長度的合計)不超過聚乙二醇的分子長。
環糊精中之被-X-NH
2修飾的羥基之數量,相對於1個環糊精,可為1~18個,宜為1~10個,為2~6個更佳。被-X-NH
2修飾的羥基之數量為上述範圍的話,促進間葉系幹細胞之脂肪分化的效果更優良,而且抑制成骨分化的效果亦更優良。被-X-NH
2修飾的羥基之數量為10個以下的話,不易產生細胞毒性。又,被-X-NH
2修飾的羥基之數量為2個以上的話,分子可動性的變化量、表面的化學組成、表面的物理化學特性(例如,接觸角、動電位(zeta potential))之變化量會變大。利用被-X-NH
2修飾的羥基之數量,可調節聚輪烷的分子可動性、表面的化學組成、表面的物理化學特性(例如,接觸角、動電位),故可製作適於促進間葉系幹細胞之脂肪分化及/或適於抑制成骨分化之分子可動性的聚輪烷表面。
X為2價有機基,且不特別限定。有機基可列舉例如:碳原子數1~10之伸烷基、碳原子數1~20之伸烯基、碳原子數1~20之伸炔基,且也可在任意位置具有側氧基,亦可插介氧基、亞胺基,也可為它們的組合。例如,組合具有側氧基的碳原子與氧基的話,會成為酯鍵,組合具有側氧基的碳原子與亞胺基的話,會成為醯胺鍵。又,具有多個氧基的伸烷基亦稱為氧伸烷基,例如包含碳原子數1~10之聚氧伸乙基、碳原子數1~10之聚氧伸丙基。有機基之具體例可列舉:亞甲基、伸乙基、伸丙基、伸丁基、伸戊基、伸己基、伸庚基、伸辛基、伸壬基、伸癸基等伸烷基;伸丙烯基、伸丁烯基、伸戊烯基、伸己烯基、伸庚烯基、伸辛烯基、伸壬烯基、伸癸烯基等伸烯基;伸丙炔基、伸丁炔基、伸戊炔基、伸己炔基、伸庚炔基、伸辛炔基、伸壬炔基、伸癸炔基等伸炔基。就具有側氧基、氧基、或亞胺基之有機基而言,宜和來自環糊精的羥基之氧原子一起形成酯鍵、碳酸酯鍵、或胺甲酸酯鍵。具有側氧基、氧基、或亞胺基之有機基可列舉例如:羰基亞甲基(-C(=O)CH
2-)等羰基伸烷基;亞甲基羰基(-CH
2C(=O)-)等伸烷基羰基;亞甲基羰基胺基(-CH
2C(=O)NH-)等伸烷基羰基胺基;羰基胺基亞甲基(-C(=O)NHCH
2-)、羰基胺基伸乙基(-C(=O)NHCH
2CH
2-)等羰基胺基伸烷基;羰基胺基伸乙基氧伸乙基(-C(=O)NHCH
2CH
2OCH
2CH
2-)等羰基胺基伸烷基氧伸烷基;羰基胺基伸乙基聚(氧伸乙基)(-C(=O)NHCH
2CH
2(OCH
2CH
2)
t-)(例如,式中,t為2~1000)等羰基胺基伸乙基聚(氧伸烷基)基;氧伸丙基(-OCH
2CH
2CH
2-)等氧伸烷基。
經修飾之環糊精宜以式(4)表示。式中,-C(=O)NH-Xa-對應於式(3)中的X,相當於X具有側氧基及亞胺基之態樣的1種。Xa為碳原子數1~9之有機基,宜為碳原子數2~9之伸烷基,為碳原子數2~8之聚氧伸乙基更佳。
[化15]
聚輪烷具備軸分子貫穿至少1個環糊精而成的結構。各環糊精可沿軸分子之主鏈方向移動,且能以軸分子之主鏈為中心旋轉。如此的結構特性稱為分子可動性。分子可動性能隨著例如環糊精之數量、對構成結構環糊精之葡萄糖進行修飾之取代基的數量而變動。
分子可動性可在培養基材的表面上施作塗佈後,使用接觸角計測定塗佈表面的靜接觸角,並使用液滴法及俘泡法(captive bubble method)來評價。分子可動性的評價也可參照例如Soft Matter, 2012, 8, 5477-5485(Ji-Hun Seo et al.),Adv. Healthcare Mater. 2015, 4, 215-222等所記載之方法。具體而言,可由以液滴法測得之水的接觸角(在大氣中之水的接觸角)與從俘泡法測得之氣泡的接觸角求出之水的接觸角(在水中之水的接觸角)之差來計算塗佈表面的接觸角遲滯(hysteresis)值。亦可和相對於經DMSO塗佈之培養基材的效果進行比較作為對照。
分子可動性可藉由變更環糊精的貫穿數及/或環糊精中之被-X-NH
2修飾的羥基的數量來調整。例如,環糊精的貫穿數為3~120的話,對於間葉系幹細胞之脂肪分化的促進及/或成骨分化的抑制可變得更理想。環糊精的貫穿數多的話,會更容易利用再沉澱來純化。
<第二實施形態>
本發明之第二實施形態係含有式(1)表示之聚輪烷的塗佈劑。本實施形態中,「式(1)表示之聚輪烷」可參照第一實施形態所說明者。
聚輪烷的含量,以塗佈劑之質量為基準,可為0.0005~5質量%,宜為0.01~1質量%,為0.02~0.5質量%更佳。
本實施形態相關之塗佈劑除了含有上述聚輪烷之外,也可含有溶劑、任意之添加劑。溶劑可列舉例如:二甲基亞碸(DMSO)、四氫呋喃(THF)、N,N-二甲基甲醯胺(DMF)、甲醇、2-丙醇、氯仿、二氯甲烷。又,如此的添加劑可列舉抗氧化劑等。
本實施形態之塗佈劑係為了塗佈於可使用作為培養基材之基板的表面而使用。可使用作為培養基材之基板可為該技術領域中具有通常知識者所習知者。基板的材質可列舉例如:玻璃、聚苯乙烯、聚丙烯、聚乙烯、聚烯烴、聚碳酸酯、丙烯酸系嵌段共聚物(BCF)等。
根據本實施形態之塗佈劑,可藉由調整構成環糊精之葡萄糖中被-X-NH
2修飾的羥基之數量,而非取決於基板本身的整體特性,來調整間葉系幹細胞之分化(脂肪分化的促進、成骨分化的抑制)。更具體而言,隨著脂肪分化進行,細胞內的脂肪滴累積量會增加。可將細胞內所累積的脂肪滴利用油紅O色素進行染色,並評價細胞內之脂肪滴累積的程度。間葉系幹細胞之脂肪分化也可利用表現脂肪分化之指標即分化標記基因(例如,PPARγ、C/EBPα、aP2)的表現量來判斷。又,在成骨分化中,隨著分化進行,骨結節會形成(也稱石灰化)。可將骨結節利用茜素紅S色素進行染色,並評價石灰化的程度。間葉系幹細胞之成骨分化也可利用表現成骨分化之指標即分化標記基因(例如,RUNX2、鹼性磷酸酶、骨鈣素(osteocalcin)、骨橋蛋白(osteopontin)、骨涎蛋白(bone sialoprotein)、I型膠原蛋白)之表現量來判斷。
聚輪烷能以實施例為參考來製造,也可如下般進行製造。將位於期望長度之聚乙二醇的兩末端之羥基轉化成脫離基(例如,鹵化、甲烷磺醯化、甲苯磺醯化),並予以苯丙胺醇(phenylalaninol)及醚化來獲得二胺。藉由將得到的二胺和環糊精進行混合來獲得假輪烷(pseudorotaxane)。此時,可藉由調整環糊精相對於1分子二胺的量來調整環糊精的貫穿數。然後,和CPADB及DMT-MM進行反應並進行封端,俾使環糊精無法從軸分子脫離後,利用可逆的加成裂解鏈轉移聚合反應(RAFT聚合反應),以錨定段的形式於兩末端導入甲基丙烯酸苄酯(相當於(甲基)丙烯酸酯結構)。
<第三實施形態>
本發明之第三實施形態係用以促進間葉系幹細胞之脂肪分化的細胞培養方法,其係包含在經含有式(1)表示之聚輪烷的組成物塗佈後之基板的表面上培養間葉系幹細胞之步驟的方法。
本實施形態中,「式(1)表示之聚輪烷」可參照第一實施形態所說明者,「含有式(1)表示之聚輪烷的組成物」可利用第二實施形態所說明之塗佈劑。
本實施形態之細胞培養方法中,係使用已塗佈含有式(1)表示之聚輪烷的組成物後之基板作為培養基材。細胞的培養係使細胞黏附於上述經塗佈後之基板的表面來實施。
培養基材係於基板施作含有式(1)表示之聚輪烷的塗佈而成者。基板可為該技術領域中具有通常知識者所習知者。基板的材質可列舉例如:玻璃、聚苯乙烯、聚丙烯、聚乙烯、聚烯烴、聚碳酸酯、丙烯酸系嵌段共聚物(BCF)等。基板也可為市售之玻璃製基板或塑膠製基板。
本實施形態之細胞培養方法係於基板上已施作之塗佈表面上,以浸漬細胞的方式投入培養基並接種培養對象之細胞,進行培養。培養基也可因應需要更換新的培養基。又,也可在使細胞分化前設置投入增殖用培養基來使接種後之細胞增殖之步驟。此時,係進行增殖直到可獲得足夠的細胞數後,再將增殖用培養基置換成分化用培養基。增殖用培養基及分化用培養基可利用該技術領域中具有通常知識者所習知的培養基。
間葉系幹細胞之脂肪分化用培養基可使用例如:PromoCell GmbH (Heidelberg, Germany)公司之間葉系幹細胞脂肪增生分化培養基(Mesenchymal Stem Cell Adipogenic Differentiation Medium2,C-28016)。
細胞培養環境可依該技術領域中具有通常知識者所習知的條件任意地設定。
間葉系幹細胞之脂肪分化意指從間葉系幹細胞朝脂肪細胞的分化。隨著脂肪分化進行,細胞內的脂肪滴累積量會增加。間葉系幹細胞之脂肪分化促進效果可利用油紅O所為之細胞染色(細胞內的脂肪滴被染色)來評價。也可藉由測定分化標記的表現量之變化來判斷。例如,脂肪分化的分化標記可列舉:PPARγ、C/EBPα、aP2。若和使用將未修飾聚輪烷(例如,後述PRX-PBzMA)塗佈於基板(玻璃製或聚苯乙烯製)而成的培養基材時比較,於統計學上顯著地促進分化,則可判斷具有間葉系幹細胞之脂肪分化促進效果。
本實施形態之細胞培養方法也可包含上述在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
含有式(1)表示之聚輪烷的組成物可塗佈於基板。塗佈的方法並無特別限定,可列舉例如:模鑄、旋塗、凹版塗佈、模塗、刀塗、棒塗、刃塗、輥塗等。理想的塗佈方法為模鑄。
<第四實施形態>
本發明之第四實施形態係用以抑制間葉系幹細胞之成骨分化的細胞培養方法,其係包含在經含有式(1)表示之聚輪烷的組成物塗佈後之基板的表面上培養間葉系幹細胞之步驟的方法。
本實施形態中,「式(1)表示之聚輪烷」可參照第一實施形態所說明者,「含有式(1)表示之聚輪烷的組成物」可利用第二實施形態所說明之塗佈劑。
本實施形態之細胞培養方法中,係使用已塗佈含有式(1)表示之聚輪烷的組成物後之基板作為培養基材。細胞的培養係使細胞黏附於上述經塗佈後之基板的表面來實施。
培養基材係於基板施作含有式(1)表示之聚輪烷的塗佈而成者。基板可為該技術領域中具有通常知識者所習知者。基板的材質可列舉例如:玻璃、聚苯乙烯、聚丙烯、聚乙烯、聚烯烴、聚碳酸酯、丙烯酸系嵌段共聚物(BCF)等。基板也可為市售之玻璃製基板或塑膠製基板。
本實施形態之細胞培養方法係於基板上已施作之塗佈表面上,以浸漬細胞的方式投入培養基並接種培養對象之細胞,進行培養。培養基也可因應需要更換新的培養基。又,也可在使細胞分化前設置投入增殖用培養基來使接種後之細胞增殖之步驟。此時,係進行增殖直到可獲得足夠的細胞數後,再將增殖用培養基置換成分化用培養基。增殖用培養基及分化用培養基可利用該技術領域中具有通常知識者所習知的培養基。
間葉系幹細胞之成骨分化用培養基可使用例如:PromoCell GmbH (Heidelberg, Germany)公司之間葉系幹細胞骨增生分化培養基(Mesenchymal Stem Cell Osteogenic Differentiation Medium,C-28013)。
細胞培養環境可依該技術領域中具有通常知識者所習知的條件任意地設定。
間葉系幹細胞之成骨分化意指從間葉系幹細胞朝成骨細胞的分化。隨著成骨分化進行,會形成骨結節(也稱石灰化)。間葉系幹細胞之成骨分化促進效果可利用茜素紅S所為之細胞染色(骨結節被染色)來評價。也可藉由測定分化標記的表現量之變化來判斷。例如,成骨分化的分化標記可列舉:RUNX2、鹼性磷酸酶、骨鈣素、骨橋蛋白、骨涎蛋白、I型膠原蛋白。若和使用將未修飾聚輪烷(例如,後述PRX-PBzMA)塗佈於基板(玻璃製或聚苯乙烯製)而成的培養基材時比較,於統計學上顯著地促進分化,則可判斷具有間葉系幹細胞之成骨分化促進效果。
本實施形態之細胞培養方法也可包含上述在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
含有式(1)表示之聚輪烷的組成物可塗佈於基板。塗佈的方法並無特別限定,可列舉例如:模鑄、旋塗、凹版塗佈、模塗、刀塗、棒塗、刃塗、輥塗等。理想的塗佈方法為模鑄。
<第五實施形態>
本發明之第五實施形態係包含經含有式(1)表示之聚輪烷的組成物塗佈後之基板的培養基材。
本實施形態中,「式(1)表示之聚輪烷」可參照第一實施形態所說明者,「含有式(1)表示之聚輪烷的組成物」可利用第二實施形態所說明之塗佈劑。又,塗佈方法可參照第三實施形態所說明之方法。
本實施形態之經塗佈後之基板(培養基材)特別適合作為用以促進間葉系幹細胞之脂肪分化及/或用以抑制成骨分化的培養基材。
根據本實施形態之培養基材,可藉由調整構成環糊精之葡萄糖中被-X-NH
2修飾的羥基之數量,而非取決於基板本身的整體特性,來調整間葉系幹細胞之分化(脂肪分化的促進及/或成骨分化的抑制)、間葉系幹細胞之增殖(促進或抑制)。
[實施例]
以下,使用實施例更詳細地說明本發明。惟,本發明不限於此。又,實施例中使用的縮寫為該技術領域中具有通常知識者所習知之慣用的縮寫,將某些縮寫的意義表示如下。
αCD:α-環糊精
CDI:羰基二咪唑
CPADB:4-氰基戊酸二硫代苯甲酸酯
DMEM:達氏改良伊氏培養基(Dulbecco’s Modified Eagle Medium)
DMF:N,N-二甲基甲醯胺
DMSO:二甲基亞碸
DMT-MM:4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基𠰌啉氯鹽
EDTA:乙二胺四乙酸
FBS:牛胎兒血清
IBMX:3-異丁基-1-甲基黃嘌呤
α-MEM:α-最低必需培養基
MeOH:甲醇
MsCl:甲磺醯氯
PBS:磷酸緩衝生理食鹽水
PEG:聚乙二醇
TCPS:細胞培養用聚苯乙烯
TEA:三乙胺
THF:四氫呋喃
1H-NMR:質子核磁共振圖譜測定
步驟1:α,ω-雙甲磺醯基聚乙二醇之合成
使數目平均分子量20000之聚乙二醇(25.0g,1.25mmol)及TEA(5.3mL,37.5mmol)溶解於無水THF(130mL)中,滴加MsCl(2.0mL,25.0mmol),並於23℃進行攪拌。5小時後,將反應液過濾,利用二乙醚使該濾液沉澱,並將沉澱物以固體形式予以回收。將得到的沉澱物於減壓下進行乾燥,獲得α,ω-雙甲磺醯基聚乙二醇(21.1g,產率:84%)。
步驟2:雙(2-胺基-3-苯基丙基)聚乙二醇之合成
將L-苯丙胺醇(1.49g,9.85mmol)及氫化鈉(0.971g,60%礦物油)在氮氣環境下,溶解於無水DMF(86mL)中。於該混合液中添加α,ω-雙甲磺醯基聚乙二醇(20.0g,0.992mmol),並於23℃進行攪拌。24小時後,將反應液過濾,利用二乙醚使該濾液沉澱,並將沉澱物以固體形式予以回收。將得到的沉澱物於減壓下進行乾燥,獲得雙(2-胺基-3-苯基丙基)聚乙二醇(11.8g,產率:59%)。
步驟3:假聚輪烷之合成
使雙(2-胺基-3-苯基丙基)聚乙二醇(10.1g,0.499mmol)溶解於水(50mL)中,添加αCD(55.2g,56.6mmol)之飽和水溶液(380mL),並於23℃進行攪拌。19小時後,藉由對反應液進行離心分離來回收沉澱物,並藉由實施冷凍乾燥9天,以粗純化物形式獲得假聚輪烷。
步驟4:聚輪烷PRX-CPADB之合成
使CPADB(5.50g,19.7mmol)及DMT-MM(5.50g,19.9mmol)溶解於甲醇(500mL)中,將上述所得到的假聚輪烷在23℃添加於反應液中,並進行攪拌。1天後,將粗純化物以甲醇進行清洗,並利用含水DMSO使其再沉澱,實施離心分離,進行冷凍乾燥9天,藉此以粉末形式獲得聚輪烷PRX-CPADB(11.9g,2步驟產率:18%)。聚輪烷PRX-CPADB的結構利用
1H-NMR(溶劑:DMSO-d
6)進行確認。又,αCD的貫穿數利用
1H-NMR(溶劑:D
2O)來確定。
1H-NMR(500 MHz, DMSO-d
6) δ 3.12-3.91 (m, PEG backbone and H2, H3, H4, H5,and H6 protons of αCD), 4.43 (m, OH6 of αCD), 4.80 (m, H1 of αCD), 5.49 (m, OH3of αCD), 5.65 (m, OH2 of αCD), 7.17 (t, aromatics of phenylalanyl group), 7.25(t, aromatics of phenylalanyl group), 7.52 (t, aromatics of CPADB group), 7.70(t, aromatics of CPADB), and 7.91 (t, aromatics of CPADB).
步驟5:參考例1之聚輪烷(PRX-PBzMA)之合成
使聚輪烷PRX-CPADB(1.50g,13.9μmol)溶解於無水DMSO(12mL)中,並於該混合液中添加甲基丙烯酸苄酯(1.71g,9.70mmol)及4,4’-偶氮雙(4-氰基戊酸)(1.55mg,5.54μmol)。利用FPT循環(Freeze-Pump-Thaw Cycle)進行脫氣後,於70℃進行攪拌。1天後,利用二乙醚使粗純化物沉澱,並於減壓下進行乾燥,獲得聚輪烷PRX-PBzMA(3.13g,產率:98%)。形成聚甲基丙烯酸苄酯結構之甲基丙烯酸苄酯單元的數量m利用
1H-NMR(溶劑:DMSO-d
6)來確定。
1H-NMR(500 MHz, DMSO-d
6) δ 0.40-0.95 (m, -CH(-
CH3)-CH2- of PBzMA), 1.43-2,10(m, -CH(-CH3)-
CH2- of PBzMA), 3.17-4.02 (m, PEG backbone and H2, H3, H4,H5, and H6 protons of αCD), 4.44 (m, OH6 of αCD), 4.80 (m, H1 of αCD), 4.86 (m,-
CH2-Ph of PBzMA), 5.49 (m, OH3 of αCD), 5.66 (m, OH2 of αCD), and 7.26(m, aromatics of PBzMA).
步驟6a:實施例1之聚輪烷(NH
2-PRX)之合成
[化17]
將聚輪烷PRX-PBzMA(200mg,0.895μmol)溶解於無水DMSO(10mL)中,然後將CDI(104mg,0.643mmol)添加於溶液中。於23℃攪拌1天後,於該溶液中添加乙二胺(0.43mL,6.43mmol),再於23℃攪拌1天。然後,將反應液利用4天的透析予以純化。將產物進行冷凍乾燥7天,以粉末形式獲得NH
2-PRX(176mg,產率73%)。胺基的數量利用
1H-NMR分析(溶劑:DMSO-d
6)來確定。
1H-NMR (500 MHz, DMSO-d
6) δ = 0.38-0.92 (m, -CH(-
CH3)-CH2- of PBzMA),1.47-2.02 (m, -CH(-CH3)-
CH2- of PBzMA), 3.00 (m, -O-CO-NH-
CH2- CH2-NH2), 3.15-4.55 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, andOH6 of α-CD), 4.86 (m, -
CH2-Ph of PBzMA, H1 of α-CD), and 7.26 (m,aromatics of PBzMA).
步驟6b:比較例1之聚輪烷(CH
3-PRX)之合成
[化18]
使聚輪烷PRX-PBzMA(0.15g,0.671μmol)溶解於無水DMSO(1.31mL)中,添加氫氧化鈉(28.7mg,0.716mmol)及碘甲烷(15μL,0.239mmol),並於23℃進行攪拌。1小時後,添加甲醇來停止反應,並進行透析3天,藉此獲得比較例1之聚輪烷(115mg,產率:75%)。甲基的數量利用
1H-NMR分析(溶劑:DMSO-d
6)來確定。
1H-NMR (500 MHz, DMSO-d
6) δ 0.40-0.95(m, -CH(-
CH3)-CH2- of PBzMA), 1.43-2.10 (m, -CH(-CH3)-
CH2- of PBzMA),3.17-4.02 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of αCD, and -OCH3 ofαCD), 4.44 (m, OH6 of αCD), 4.57-5.10 (m, H1 of αCD and -
CH2-Ph ofPBzMA), 5.49 (m, OH3 ofαCD), 5.64 (m, OH2 ofαCD), and 7.26 (m, aromatics ofPBzMA).
步驟6c:比較例2之聚輪烷(OH-PRX)之合成
[化19]
將聚輪烷PRX-PBzMA(200mg,0.895μmol)溶解於無水DMSO(10mL)中,並將CDI(130mg,0.804mmol)添加於溶液中。於23℃攪拌1天後,將2-(2-胺基乙氧基)乙醇(0.80mL,8.04mmol)添加於溶液中,再於23℃攪拌1天。然後,將反應液利用4天的透析予以純化。將產物進行冷凍乾燥7天,以粉末形式獲得OH-PRX(194mg,產率74%)。OH基的數量利用
1H-NMR分析(溶劑:DMSO-d
6)來確定。
1H-NMR (500 MHz,DMSO-d
6) δ 0.42-0.95 (m, -CH(-
CH3)-CH2- of PBzMA), 1.45-2.02(m, -CH(-CH3)-
CH2- of PBzMA), 3.14 (m, -O-CO-NH-
CH2-CH2-O-CH2- CH2-OH),3.17-4.70 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, OH6 ofα-CD, and -O-CO-NH-CH2-
CH2-O-
CH2-
CH2-OH), 4.86 (m, -
CH2-Phof PBzMA, H1 of α-CD), and 7.26 (m, aromatics of PBzMAH).
步驟6d:比較例3之聚輪烷(SO
3H-PRX)之合成
[化20]
將聚輪烷PRX-PBzMA(150mg,0.671μmol)溶解於無水DMSO(3.0mL)中,其後,將NaOH粉末(65.1mg,1.63mmol)及1,3-丙烷磺內酯(66.3mg,0.543mmol)添加於溶液中。於23℃攪拌1天後,進行透析純化5天。將產物進行冷凍乾燥7天,以粉末形式獲得SO
3H-PRX(155mg,產率68%)。磺酸基(-SO
3H)的數量利用
1H-NMR分析(溶劑:DMSO-d
6)來確定。
1H-NMR (500 MHz,DMSO-d
6) δ = 0.33-0.95 (m, -CH(-
CH3)-CH2- of PBzMA),1.41-2.12 (m, -CH(-CH3)-
CH2- of PBzMA and -O-CH2-
CH2-CH2- SO3Na),2.92-4.23 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, and -O-
CH2-CH2-CH2-SO3Na),4.86 (m, -
CH2-Ph of PBzMA, H1 of α-CD), 7.26 (m, aromatics of PBzMA).
得到的聚輪烷之資料如表1所示。圖1係將參考例1、實施例1、比較例1~3之聚輪烷之各
1H-NMR圖譜進行比較的圖。各圖譜中之特徵的峰部以灰色表示。括弧內的數字表示每1個環糊精的官能基修飾數。
[表1]
PEG部分的數目平均分子量 | 聚甲基丙烯酸苄酯的數目平均分子量 | αCD的貫穿數 | 被官能基化的羥基數 | |
參考例1 | 20000 | 115200 | 89.8 | 0(0) |
實施例1 | 20000 | 115200 | 89.8 | 310(3.5) |
比較例1 | 20000 | 115200 | 89.8 | 312(3.5) |
比較例2 | 20000 | 115200 | 89.8 | 392(4.4) |
比較例3 | 20000 | 115200 | 89.8 | 568(6.3) |
將得到的聚輪烷溶解於DMSO中,製造塗佈劑。將得到的塗佈劑模鑄於TCPS。
2.表面化學組成的分析
(1)表面的元素組成
利用使用了30keV之Bi3
++一次離子的TOF-SIMS(PHI NanoTOF II,ULVAC-PHI)來分析聚輪烷表面的化學組成。分析視野為100×100μm。表面的元素組成係利用使用了放射具有1486.6eV之能量的單色化之X射線之X射線光電子分光(Al-Kα,Thermo Fisher Scientific, East Grinstead)來確定。分析區域的直徑為400μm,元素組成由各表面之3處的平均來計算。
TOF-SIMS測定而得的質譜圖如圖2~3所示。TCPS以外的表面觀測到m/z=85之特性峰部。該峰部教示甲基丙烯酸酯基(C
4H
5O
2 -)的存在,據推測TCPS表面係經含有PBzMA結構之三嵌段共聚物塗佈。又,對於實施例1及比較例2之表面,檢測到教示含有N之基(CNO
-)的存在之於m/z=42的特徵峰部。由表2所示之XPS分析的結果可知,氮及氧的元素組成在NH
2-PRX表面(實施例1)及OH-PRX(比較例2)上,顯著地高於其它表面上。
[表2]
元素(原子%) | ||||
C1s | O1s | N1s | S2p | |
參考例1 | 95.8±0.3 | 4.0±0.5 | 0.2±0.1 | 0.0±0.0 |
實施例1 | 78.8±0.2 | 19.1±0.3 | 2.1±0.1 | 0.0±0.0 |
比較例1 | 94.0±2.4 | 5.8±2.3 | 0.3±0.1 | 0.0±0.0 |
比較例2 | 77.7±0.4 | 20.4±0.4 | 1.9±0.0 | 0.0±0.0 |
比較例3 | 92.6±2.7 | 6.5±2.3 | 0.2±0.2 | 0.3±0.2 |
(2)接觸角及動電位的測定
於細胞培養用聚苯乙烯(TCPS)之表面施作含有實施例1及比較例1~3之聚輪烷的塗佈。使用接觸角計(商品名:DM-501,協和界面科學股份有限公司製)及軟體,以液滴法及俘泡法兩種測定塗佈表面的靜接觸角。各表面之接觸角遲滯值係由以液滴法測得之水的接觸角(在大氣中之水的接觸角)及從俘泡法測得之氣泡的接觸角求出之水的接觸角(在水中之水的接觸角)之差來計算。所有的測定值皆為4個不同的表面所得之值,並記錄各表面之3個不同點的平均值。已知接觸角遲滯的差即表示表面間之分子可動性的差。
參考例1、實施例1及比較例1~3之聚輪烷表面的接觸角及接觸角遲滯如表3所示。聚輪烷表面在大氣中之水的接觸角落在80~100°之範圍,並確認因聚輪烷塗佈而TCPS表面(接觸角為74°)的潤濕性發生變化。實施例1及比較例1~3之聚輪烷表面在大氣中之水的接觸角比起參考例1之聚輪烷表面的接觸角稍微小,聚輪烷表面間並無顯著差異。另一方面,在水中使用俘泡法而測得的在水中之水的接觸角則表現不同的傾向,於聚輪烷表面間觀察到顯著差異。
聚輪烷表面具有來自αCD及聚乙二醇鏈間之互鎖(interlock)結構的分子可動性,並以水合狀態展現獨特的行為。例如已知就聚輪烷表面在水合時的分子可動性而言,(1)由伴隨散逸之使用石英晶體微天平於水中測得之散逸能量損失(QCM-D)所求得的結果、及(2)利用空氣中之水的接觸角及由水中之氣泡的接觸角求出之水的接觸角之差所測得之接觸角遲滯的結果間存在關聯性。在使用了實施例1及比較例1~3之各塗佈表面中,由於因表面官能基的種類而接觸角遲滯之值會變化,可認為環糊精之羥基的官能基修飾用於調整分子可動性係為有效。
又,使用電泳光散射分光計(ELSZ-2,大塚電子),並利用平板樣本用之石英流動型比色管測定聚輪烷表面的動電位。使用於10mM PBS中之監測粒子(大塚電子)來分析表面上的電氣滲透之移動度,並計算表面上的動電位。所有的測定係於3個不同的表面實施。
參考例1、實施例1及比較例1~3之聚輪烷表面的動電位如表3所示。實施例1之聚輪烷表面的負電荷最小,比較例3在所有表面之中帶最多負電。胺基在pH7.4應帶正電,但實施例1之聚輪烷表面帶負電。據認為此係因塗佈的基底之基板(TCPS)的負電荷以及未修飾PRX表面的負電荷所致。
[表3]
在大氣中之水的接觸角(θ) | 在水中之水的接觸角(θ) | 接觸角遲滯(θ) | 動電位(mV) | |
參考例1 | 93.8±1.3 | 75.5±1.5 | 18.3±1.1 | -24.6±2.8 |
實施例1 | 80.8±2.1 | 43.0±1.4 | 37.8±2.8 | -9.9±0.7 |
比較例1 | 85.0±1.7 | 60.0±2.4 | 25.0±3.9 | -29.3±2.8 |
比較例2 | 83.5±1.1 | 52.9±4.0 | 30.7±5.1 | -16.3±6.3 |
比較例3 | 86.5±2.7 | 22.2±2.0 | 64.3±3.8 | -33.6±3.4 |
(3)對聚輪烷表面之纖連蛋白吸附(微量BCA蛋白測定,Micro BCA Assay)
為了檢查對聚輪烷表面之纖連蛋白吸附,於參考例1、實施例1及比較例1~3之各表面,將人類纖連蛋白之PBS溶液(100μg/mL)於37℃培養3小時。將各孔盤以PBS清洗3次,去除未吸附纖連蛋白。依循J. Biomater. Sci. Polym. Ed., 2017, 28, 986-999.或ACSBiomater. Sci. Eng., 2018, 4, 1591-1597.所記載之方法,添加5%SDS及0.1N NaOH水溶液,並於37℃培養1小時,藉此萃取已吸附之纖連蛋白。纖連蛋白濃度係依循製造業者之指示,和人類纖連蛋白標準品一起使用蛋白質分析套組(Micro BCA(商標),Thermo Scientific製)進行測定。
結果如圖4所示。在實施例1的表面吸附最多纖連蛋白。據認為纖連蛋白帶負電,並因為和帶正電之胺基的靜電交互作用而吸附較多纖連蛋白。比較例1~3之聚輪烷表面的纖連蛋白吸附量,相對於參考例1並無顯著差異。
2.聚輪烷表面的細胞反饋
(1)聚輪烷表面上之人類間葉系幹細胞(hMSC)黏附及增殖
為了評價各聚輪烷表面上之hMSC的初期黏附及增殖,將細胞以6.0×10
3細胞/cm
2之濃度接種於各表面上,於含有5%CO
2之加濕環境中,使用hMSC增殖用培養基BulletKit在37℃培養3天。細胞的形態使用相位差顯微鏡(BZ-X700,KEYENCE)進行觀察,並將黏附的細胞利用使用了胰蛋白酶/EDTA溶液之處理而從基板剝離,使用血球計數器每隔1天測定黏附於各表面之hMSC的數量。
結果如圖5所示。接種後第1天,於各聚輪烷表面間觀察到細胞密度並無顯著差異。接種第3天,實施例1之表面上的細胞密度最低。
(2)聚輪烷表面上之hMSC的成骨分化
為了誘導成骨分化(骨形成分化),於各聚輪烷表面將hMSC以2.4×10
4細胞/cm
2之密度進行接種,使用hMSC增殖用培養基BulletKit,直到細胞之密度達到過度簇集(excessive confluent)為止,於含有5%CO
2之加濕環境下,在37℃培養5天。將增殖用培養基更換成成骨分化用培養基,並將細胞培養14天。培養基每3~4天更換成新鮮的培養基。成骨分化用培養基使用PromoCell GmbH (Heidelberg, Germany)公司之間葉系幹細胞骨增生分化培養基(Mesenchymal Stem Cell Osteogenic Differentiation Medium,C-28013)。
分化誘導之14天後,將細胞以茜素紅S進行染色,並評價hMSC之石灰化的程度。藉由將細胞以PBS清洗2次,並使用4%多聚甲醛溶液於23℃處理10分鐘來固定。將細胞以MilliQ水清洗2次,並使用茜素紅S溶液於23℃染色10分鐘。去除染色液後,將細胞以MilliQ水清洗4次並用顯微鏡進行觀察。染色面積使用ImageJ軟體進行計算。所有的測定值係於3個不同的表面所得的值,並令各表面之4個不同處之平均值為各表面之平均值。
結果如圖6所示。骨形成分化之誘導後,細胞形態由細長形狀變化成正方形的形狀,細胞密度尤其在比較例3之表面上有增加。為了評價在各表面上培養的hMSC之骨結節形成,於成骨分化誘導後第14天使用茜素紅S來染色鈣結節。圖7中,(A)為利用茜素紅S所為之染色照片、及(B)為將各聚輪烷表面的染色面積進行比較之圖表。染色面積觀察到顯著差異,帶最多負電的表面(比較例3)最大,最不帶負電之表面(實施例1)最小,其它表面(參考例1、比較例1~2)則為中間程度。
(3)聚輪烷表面上之hMSC的脂肪分化
為了誘導脂肪分化,於各聚輪烷表面將hMSC以1.0×10
4細胞/cm
2之密度進行接種,使用hMSC增殖用培養基BulletKit,於含有5%CO
2之加濕環境下,在37℃培養5天。將增殖用培養基更換成脂肪分化用培養基,並將細胞培養15天。培養基每3~4天更換成新鮮的培養基。脂肪分化用培養基使用PromoCell GmbH (Heidelberg, Germany)公司之間葉系幹細胞脂肪增生分化培養基(Mesenchymal Stem Cell Adipogenic Differentiation Medium2,C-28016)。
分化誘導之15天後,將細胞以油紅O進行染色,並評價細胞內之脂肪滴累積。藉由將細胞以PBS清洗2次,並使用4%多聚甲醛溶液於23℃處理10分鐘來固定。將細胞以PBS清洗2次,並以60%之2-丙醇清洗1次。將細胞於23℃使用油紅O溶液染色20分鐘,並以60%之2-丙醇清洗1次。最後將細胞以PBS清洗2次,並使用顯微鏡於PBS中進行觀察。染色面積使用ImageJ軟體進行計算。所有的測定值係於3個不同的表面所得的值,並令各表面之3個不同處之平均值為各表面之平均值。
結果如圖8所示。伴隨脂肪分化之細胞內的脂肪滴形成在全部的表面皆可觀察到,並隨著培養期間觀察到脂肪滴的增加。圖9中,(A)為利用油紅O所為之染色照片、及(B)為將各聚輪烷表面中的染色面積進行比較之圖表。實施例1及比較例2~3之表面上的染色面積比起參考例1及比較例1之表面上的染色面積大。觀察到利用油紅O所為之染色面積隨著接觸角遲滯的增加而增加之傾向。由於接觸角遲滯的差即表示表面之分子可動性的差,故據認為尤其實施例1及比較例2~3之聚輪烷表面的分子可動性更適於促進脂肪分化。
[圖1]係參考例1、實施例1及比較例1~3之
1H-NMR圖譜的比較圖。
[圖2]係參考例1、比較例1~2之利用TOF-SIMS測定而得到的質譜圖。
[圖3]係實施例1、比較例3、TCPS之利用TOF-SIMS測定而得到的質譜圖。
[圖4]係顯示參考例1、實施例1及比較例1~3之纖連蛋白吸附量的圖表。
[圖5]係顯示參考例1、實施例1及比較例1~3之細胞密度的圖表。
[圖6]係拍攝使用參考例1、實施例1及比較例1~3來誘導成骨分化後之細胞的顯微鏡照片(比例尺為100μm)。
[圖7](A)係將使用參考例1、實施例1及比較例1~3來誘導成骨分化後之細胞以茜素紅S染色而成的照片(比例尺為200μm),(B)係染色面積的比較圖表。
[圖8]係拍攝參考例1、實施例1及比較例1~3之誘導脂肪分化後之細胞的顯微鏡照片(比例尺為100μm)。
[圖9](A)係將使用參考例1、實施例1及比較例1~3來誘導脂肪分化後之細胞以油紅O染色而成的照片(比例尺為100μm),(B)係染色面積的比較圖表。
Claims (15)
- 如請求項2之塗佈劑,其中,該聚輪烷含有1~18個羥基被-X-NH 2表示之基修飾而成的環糊精。
- 如請求項2或3之塗佈劑,其中,該聚輪烷中,環糊精的貫穿數為3~220個。
- 如請求項5之細胞培養方法,其包含在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
- 如請求項5或6之細胞培養方法,其中,該聚輪烷含有1~18個羥基被-X-NH 2表示之基修飾而成的環糊精。
- 如請求項5至7中任一項之細胞培養方法,其中,該聚輪烷中,環糊精的貫穿數為3~220個。
- 如請求項9之細胞培養方法,其包含在基板的表面上塗佈含有式(1)表示之聚輪烷的組成物之步驟。
- 如請求項9或10之細胞培養方法,其中,該聚輪烷含有1~18個羥基被-X-NH 2表示之基修飾而成的環糊精。
- 如請求項9至11中任一項之細胞培養方法,其中,該聚輪烷中,環糊精的貫穿數為3~220個。
- 如請求項13之培養基材,其中,該聚輪烷含有1~18個羥基被-X-NH 2表示之基修飾而成的環糊精。
- 如請求項13或14之培養基材,其中,該聚輪烷中,環糊精的貫穿數為3~200個。
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WO2017191827A1 (ja) * | 2016-05-02 | 2017-11-09 | 国立大学法人 東京医科歯科大学 | 内部分解型ポリロタキサンおよびその合成方法 |
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2021
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KR20230091942A (ko) | 2023-06-23 |
EP4253446A1 (en) | 2023-10-04 |
AU2021385715A1 (en) | 2023-06-29 |
CN116507665A (zh) | 2023-07-28 |
CA3203148A1 (en) | 2022-06-02 |
AU2021385715A9 (en) | 2024-09-12 |
EP4253446A4 (en) | 2024-05-15 |
JPWO2022113940A1 (zh) | 2022-06-02 |
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US20240240048A1 (en) | 2024-07-18 |
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