TWI484983B - 應用於呼吸上皮細胞之敷料 - Google Patents
應用於呼吸上皮細胞之敷料 Download PDFInfo
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- TWI484983B TWI484983B TW101145941A TW101145941A TWI484983B TW I484983 B TWI484983 B TW I484983B TW 101145941 A TW101145941 A TW 101145941A TW 101145941 A TW101145941 A TW 101145941A TW I484983 B TWI484983 B TW I484983B
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- acid
- hyaluronic acid
- vitamin
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- chitosan
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- 230000000241 respiratory effect Effects 0.000 title description 16
- 210000002919 epithelial cell Anatomy 0.000 title description 12
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 30
- 229930002330 retinoic acid Natural products 0.000 claims description 30
- 239000000203 mixture Substances 0.000 claims description 27
- 229920002674 hyaluronan Polymers 0.000 claims description 23
- 229960003160 hyaluronic acid Drugs 0.000 claims description 23
- 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 group 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 claims description 19
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F13/00—Bandages or dressings; Absorbent pads
- A61F13/00051—Accessories for dressings
- A61F13/00063—Accessories for dressings comprising medicaments or additives, e.g. odor control, PH control, debriding, antimicrobic
-
- A61F13/01012—
-
- A61F13/01017—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
Description
本發明係關於一種用於呼吸道之敷料,特別是一種可調控呼吸道上皮細胞之敷料。
呼吸上皮為黏液纖毛組織,為呼吸道抵禦外來物質(例如微生物)之第一道防線。鼻部呼吸上皮缺損之患者之病症包括:鼻腦脊髓液漏會產生頭痛、腦部感染;鼻中膈缺損會產生發炎、結痂增生、出血、呼吸哨音等症狀,臨床治療多採用修補手術,常見修補術式例如黏膜皮瓣、側邊鼻皮瓣、顳骨筋膜自體皮瓣等,然而,前述修補手術時常伴隨移植部位病變、組織攣縮、殘存原組織特性等問題。
近年來,業界發展出以生物相容性材料作為移植物,大多採用膠原蛋白做為原料,但缺點為膠原蛋白易降解、機械強度不足、價格昂貴,在臨床應用上造成限制。
因此,可應用於呼吸道之生醫材料仍有其需求。
本發明係提供一種用於呼吸道之敷料,包括:一具有生物相容性之高分子聚合物,以該敷料總體積為基準,佔99%以上;及維生素A酸(retinoic acid),以該敷料總體積為基準,佔1%以下。
本發明亦提供一種調控呼吸道上皮細胞之生長之方法,係包括:提供一種包括生物相容性高分子聚合物及維生素A酸之混合物,其中,以該混合物總體積為基準,該高分子聚合物佔99%以上且該維生素A酸佔1%以下;以及使該呼吸道上皮細胞與該混合物接觸達預定時間。
於本發明之敷料中,以該敷料總體積為基準,該具有生物相容性之高分子聚合物係佔99%以上且未達100%,例如,99.1%、99.3%、99.5%、99.7%、99.9%、99.99%,或為前述任兩點之間之數值;又,該維生素A酸係佔1%以下,例如0.9%、0.7%、0.5%、0.3%、0.1%、0.05%、0.01%,或為前述任兩點之間之數值。
本發明之敷料具有維生素A酸之持釋作用,每份敷料能夠控制持續並穩定的釋放維生素A酸長達數天,例如可達約7天至10天。
本發明之敷料可為固態、液態、膠態,視需要可進一步搭配其他載體使用,主要應用於呼吸道,尤其是應用於呼吸道上皮細胞,例如作為創傷修復敷料、貼片、填補材料、移植物等使用。於應用於呼吸道創傷修復之實施例中,該敷料可直接覆蓋於傷口處,該高分子聚合物可阻隔傷口與外在環境之接觸,並對該傷口處提供一封閉的濕潤環境,而維生素A酸可由該敷料持續且穩定地釋放至傷口處,藉此促進呼吸道上皮細胞的修復、增殖、及分化,有利於傷口癒合。
因此,本發明亦提供一種調控呼吸道上皮細胞之生長之方法,係包括:提供一種包括生物相容性高分子聚合物及維生素A酸之混合物,其中,以該混合物總體積為基準,該高分子聚合物佔99%以上且該維生素A酸佔1%以下;以及使該呼吸道上皮細胞與該混合物接觸達預定時間,以使該呼吸道上皮細胞與該混合物貼附、增殖、及分化。
該呼吸道上皮細胞經該混合物/敷料處理後,能有效分化成纖毛構造。
於該方法中,該混合物係包括,以該混合物總體積為基準,該高分子聚合物佔99%以上且該維生素A酸佔1%以下。
於一較佳實施例中,該高分子聚合物佔99.9%以上且該維生素A酸佔0.1%以下。
於一實施例中,該呼吸道上皮細胞與該混合物接觸係達一定時間以上,以使該混合物釋出足夠量之維生素A酸作用於該細胞。於一實施例中,呼吸道上皮細胞與混合物的接觸時間可達4小時以上,例如:8小時、16小時、1天、3天、7天、10天等。
於一實施例中,該高分子聚合物係選自透明質酸(hyaluronic acid)、幾丁聚醣(chitosan)、膠原蛋白(collagen)、或其任意之組合。於實施例中,前述透明質酸、幾丁聚醣、或膠原蛋白亦可為其衍生物或經改質(modified)者。
透明質酸係指由雙糖(D-葡萄糖醛酸及N-乙醯葡糖胺)構成之單元重複組成之多醣類,於一實施例中,該透明質酸之分子量可為5 KDa至20,000 KDa。
於另一實施例中,透明質酸可為,例如透明質酸酯類化合物,包括透明質酸乙酯、透明質酸丙酯、透明質酸芳香酯(如透明質酸苯酯化合物)、透明質酸丙烯酸酯、透明質酸羧酸酯等;經交聯處理之透明質酸,例如以二乙烯基碸(DVS)、碳化二亞胺(EDC)、甲醛、環氧化物、金屬陽離子等之交聯劑處理者;經改質或經接枝之透明質酸,包括以甲基丙烯酸縮水甘油改質之透明質酸、聚乙二醇-聚丙二醇共聚物接枝之透明質酸等;可單獨或組合使用。於實施例中,以透明質酸酯類為較佳。
幾丁聚醣係指主要由葡萄糖胺所構成之多醣類。於一實施例中,幾丁聚醣可為,例如幾丁聚醣之烷基衍生物,如乙基、丁基、辛基、十六烷基、羥乙基等;幾丁聚醣單醣衍生物,如與葡萄糖、果糖、半乳糖、葡萄醣胺等單醣共價鍵結者;幾丁聚醣雙醣衍生物,如與乳糖、麥芽糖、纖維雙醣等雙醣共價鍵結者;可單獨或組合使用。
於實施例中,膠原蛋白之類型並未特別限制,可為第I型(type I)、第II型、第III型、第IV型等。於實施例中,膠原
蛋白之應用型態並未特別限制,可為纖維狀、管狀、多孔狀、薄膜、海綿狀、注射劑型等。
於一實施例中,該敷料所包含之維生素A酸之濃度可為1 M至1×10-10
M。維生素A酸之濃度為1×10-3
M至1×10-7
M為較佳,濃度為6×10-5
M至1×10-6
M為更佳。
於本發明中,維生素A酸由該敷料釋出之比例係所經時間之函數,且通常不受維生素A酸之初始濃度之影響,因此,由於前述釋出作用,本發明之敷料可達到動態調控的功效。
以下以實例詳細說明本發明。
實例
1.製備維生素A酸-透明質酸衍生物之混合物(RA-HAm)
所有步驟均於暗室進行。取180 mg之透明質酸苯酯化合物(HYAFF)(購自Fidia Advanced Biomaterials,Italy)於室溫下溶解於1 ml之二甲亞碸(DMSO)中。於上述溶液中添加10 μl之維生素A酸(於乙醇中,濃度10-3
M)並混合均勻。將該混合物以150 μl/cm2
之量塗佈於培養盤表面。加入HYAFF溶液體積100倍之乙醇,用以沈澱HYAFF。小心地將該聚合物膜取下,並於室溫、暗室、真空下乾燥12小時。於使用前以磷酸緩衝食鹽水(PBS)潤洗該膜。
2. RA釋出測試
將前述所製備得RA-HAm置於細胞培養插入皿(Transwell insert)中,且每日更換培養基。將RA-HAm溶解於500μl之DMSO後以ELISA測量儀測定波長380 nm之吸光值,藉此定量RA-HAm中維生素A酸的殘留量。同時亦測量培養基中的維生素A酸的量。所有步驟均於暗室進行。
結果如第1圖所示,(A)為RA-HAm中維生素A酸的殘留
量,維生素A酸的起始濃度為420μM,第1天有較大量之釋出,接著釋出速率為緩慢且持續,可維持釋出達約7天。(B)為培養基中的維生素A酸累積量,於第7天之累積率約87%。顯示本發明之敷料可穩定且持續的釋放維生素A酸。
3.人類呼吸上皮細胞之分離及培養
人類呼吸上皮細胞之分離及培養係參照文獻(Huang TW et al.,Acta Biomater(2010),6:1191-9及Huang TW et al.,Laryngoscope(2009),119:2066-70)。由接受鼻中膈鼻道成形術之病患取得下鼻甲(human nasal inferior turbinate)。將取得之組織以0.5%鏈黴蛋白酶(Pronase)(購自Sigma-Aldrich,USA)於添加抗生素(青黴素/鏈黴素)之培養基DMEM:F12為1:1之混合物中,4℃下,處理16-20小時。將細胞懸浮液過濾以移除細胞團及殘渣,並進行離心,接著將細胞重新溶散於添加抗生素之培養基DMEM/F12:BEGM(購自Clonetics Corp.,CA)為1:1之混合物中。將細胞以37℃預培養於塑膠培養盤上1小時,藉由分化貼附作用可移除纖維母細胞。接著,收集懸浮液中的細胞,並以培養基重新溶散成105
細胞/ml之濃度。取1.5 ml所得細胞懸浮液接種於RA-HAm上,置於細胞培養插入皿中,並於基底側(basolateral side)注入2.6 ml之培養基。於37℃、含5%二氧化碳之氛圍下進行培養。細胞達緻密(confluence)前以浸漬培養,且於培養48小時後首次更換培養基,之後每隔一天更換。待緻密後,藉由移除頂部培養基而製造氣-液界面(ALI),並由基底側部分注入培養基。
4.細胞檢測
(1)表形檢測
以光學顯微鏡觀察未處理之活細胞。又,分別以PBS及0.1 M之二甲胂緩衝液(cacodylate buffer)潤洗培養株,於含有2.5%戊二醛之0.05 M二甲胂緩衝液中固定1小時,以0.1 M
二甲胂緩衝液潤洗1小時,再以含有1% OsO4
之0.05 M二甲胂緩衝液後固定1小時。以梯度式乙醇將樣本脫水,並利用液態二氧化碳以臨界點乾燥法進行乾燥。接著,濺鍍厚度約25 nm之金,再將樣本置於掃瞄式電子顯微鏡下檢測。
第2圖顯示呼吸上皮細胞(RECs)達緻密程度時之表形,其中,第2(A)圖為HAm組,第2(B)圖為RA-HAm組,均呈上皮細胞狀(epithelial-like)。
(2)細胞活性檢測
以MTT法測量各時間點之細胞活性,以測定細胞增殖率。結果如第3圖所示,甲臢(formazan)吸收量可直接反應細胞數量,在RA-HAm組及HAm組中均顯示了細胞數量持續增加,雖然RA-HAm組於第1天後並未迅速的增加,但在第3天後兩組的細胞增殖率為相似,且兩組的呼吸上皮細胞均需時約7-8天可達緻密程度。
(3)細胞分化檢測
如前述步驟所述,於細胞達緻密程度後,製造ALI。
以掃瞄式電子顯微鏡檢測RA-HAm組及HAm組之細胞表形,如第4圖所示,呈現極顯著之差異。4(B)圖為RA-HAm組,於ALI第21天,RA-HAm敷料之上表面可觀察到已有大量的成熟纖毛細胞及微絨毛產生;然而,4(A)圖之HAm組僅形成叢集之擬纖毛結構。
可由MUC5AC蛋白質之表現評估黏膜分化作用,MUC5AC為呼吸道上皮的主要黏蛋白。取ALI第21天之RA-HAm及HAm組之呼吸上皮細胞,進行MUC5AC表現之檢測。結果如第5圖之西方墨點法結果所示,RA-HAm組之表現量高於HAm組,以肌動蛋白(actin)為基準進行量化,HAm組之MUC5AC表現量指數為0.17±0.04,而RA-HAm組為0.34±0.07,確實具有顯著差別。
另外,在黏膜纖毛系統中,維持水平衡與纖毛液量對於該系統功能至為重要。於呼吸道黏膜纖毛系統中,可藉由AQP5
蛋白質之表現量來評估。取達緻密程度後第21天之RA-HAm及HAm組之呼吸上皮細胞,進行AQP5表現量之檢測。結果如第6圖之西方墨點法結果所示,RA-HAm組之表現量高於HAm組,以肌動蛋白(actin)為基準進行量化,HAm組之AQP5表現量指數為0.2±0.02,而RA-HAm組為0.38±0.04,確實具有顯著差別。
綜合上述結果,證實本發明之具有生物相容性之高分子聚合物及維生素A酸之混合物能夠持續釋放維生素A酸至環境中,並可有效的幫助呼吸上皮細胞增殖並分化成纖毛細胞,同時可有助於促進並維持鼻黏膜纖毛系統的功能,確實適合作為呼吸道之敷料。
上述特定實施例之內容係為了詳細說明本發明,然而,該等實施例係僅用於說明,並非意欲限制本發明。熟習本領域之技藝者可理解,在不悖離後附申請專利範圍所界定之範疇下針對本發明所進行之各種變化或修改係落入本發明之一部分。
第1A圖顯示RA-HAm中維生素A酸的殘留量。
第1B圖為培養基中的維生素A酸累積量。
第2圖顯示呼吸上皮細胞(RECs)達緻密程度時之表形,其中,(A)為HAm組,(B)為RA-HAm組。
第3圖為細胞活性檢測結果。
第4圖為掃瞄式電子顯微鏡影像,其中,(A)為HAm組,(B)為RA-HAm組。
第5圖顯示MUC5AC蛋白質之表現。
第6圖顯示AQP5蛋白質之表現。
Claims (6)
- 一種用於呼吸道之敷料,包括:一具有生物相容性之高分子聚合物,以該敷料總體積為基準,佔99%以上;其中,該高分子聚合物係選自透明質酸、幾丁聚醣、膠原蛋白、或其任意之組合;及維生素A酸,以該敷料總體積為基準,佔1%以下;其中,該維生素A酸之濃度為6×10-5 M至1×10-6 M;其中,該敷料具有維生素A酸之持釋作用。
- 如申請專利範圍第1項之敷料,其中,該透明質酸係選自由透明質酸、透明質酸酯類化合物、經交聯處理之透明質酸、經改質之透明質酸、經接枝之透明質酸所成群組之一種或多種。
- 如申請專利範圍第2項之敷料,其中,該透明質酸酯類化合物包括透明質酸乙酯、透明質酸丙酯、透明質酸芳香酯、透明質酸丙烯酸酯、透明質酸羧酸酯、或其任意之組合。
- 如申請專利範圍第1項之敷料,其中,該幾丁聚醣係選自由幾丁聚醣、幾丁聚醣之烷基衍生物、幾丁聚醣單醣衍生物、及幾丁聚醣雙醣衍生物所成群組之一種或多種。
- 一種用於調控呼吸道上皮細胞之生長之組成物,係包括:一生物相容性高分子聚合物及維生素A酸之混合物,其中,以該混合物總體積為基準,該高分子聚合物佔99%以上且該維生素A酸佔1%以下;該高分子聚合物係選自透明質酸、幾丁聚醣、膠原蛋白、或其任意之組合;該維生素A酸之濃度為6×10-5 M至1×10-6 M;其中,該組成物與該呼吸道上皮細胞接觸達4小時以上,該維生素A酸係由該混合物中持續釋放。
- 如申請專利範圍第5項之組成物,其中,該呼吸道上皮細胞係與該混合物貼附,並增殖及分化。
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US4851521A (en) * | 1985-07-08 | 1989-07-25 | Fidia, S.P.A. | Esters of hyaluronic acid |
US5955109A (en) * | 1985-12-18 | 1999-09-21 | Advanced Polymer Systems, Inc. | Methods and compositions for topical delivery of retinoic acid |
US20030206958A1 (en) * | 2000-12-22 | 2003-11-06 | Cattaneo Maurizio V. | Chitosan biopolymer for the topical delivery of active agents |
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