TWI472348B - 可通過血腦障壁之胜肽及包含此胜肽之傳輸系統 - Google Patents
可通過血腦障壁之胜肽及包含此胜肽之傳輸系統 Download PDFInfo
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Description
本發明係有關於一種胜肽,特別是有關於一種可通過血腦障壁之胜肽及包含此胜肽之傳輸系統。
血腦障壁(blood brain barrier,BBB)是由腦內皮細胞(brain endothelial cells)所構成,由於細胞之間的緻密接合(tight junctions),可阻擋例如毒素(toxins)的外來物質通過血腦障壁。然而,親脂性或低分子量分子可藉由被動擴散(passive diffusion)通過血腦障壁。
某些用來治療腦或神經疾病例如親水性蛋白質藥物的活性化合物由於其高分子量或親水性質致無法藉由被動擴散通過腦組織。
因此,已開始進行藥物結構修飾的研究,以增加藥物親脂性,容許各種活性化合物通過血腦障壁。其他容許親水性或巨分子藥物通過血腦障壁的方法包括例如以吸收為媒介的細胞傳輸(absorption-mediated transcytosis,AMT),其容許正電荷載體藉由電荷吸收通過血腦障壁,以載體為媒介的細胞傳輸(carrier-mediated transcytosis,CMT),其容許例如鈉或鉀的親水性金屬離子、二胜肽、三胜肽或葡萄糖藉由傳輸子(transporters)通過血腦障壁,以及以受體為媒介的細胞傳輸(receptor-mediated transcytosis,RMT),其容許例如胰島素、運鐵蛋白(transferrin)或低密度脂蛋白(low-density lipoprotein,LDL)的巨分子藉由細胞傳輸通過血腦障壁。
本發明之一實施例,提供一種經分離之胜肽,包括:一胺基酸序列SEQ ID NO: 1。
本發明之一實施例,提供一傳輸系統,包括:一載體,具有一表面;一藥物或一染料,包覆於該載體內;以及一經分離之胜肽,包括一胺基酸序列SEQ ID NO: 1,接枝於該載體之該表面。
本發明提供一種新穎的胜肽配體(BP02,具有一胺基酸序列SEQ ID NO: 1 KYLAYPDSVHIW),可標記並通過血腦障壁(BBB)。本發明接枝有上述胜肽配體包覆藥物的載體當進入腦部時可避免藥物遭受破壞。
為讓本發明之上述目的、特徵及優點能更明顯易懂,下文特舉一較佳實施例,作詳細說明如下:
本發明之一實施例,提供一種經分離之胜肽,包括:一胺基酸序列SEQ ID NO: 1。
上述胜肽可包括一由12個胺基酸殘基所組成的胺基酸序列KYLAYPDSVHIW(SEQ ID NO: 1)。值得注意的是,上述胜肽於體外(in vitro
)與體內(in vivo
)皆可結合至血腦障壁(BBB)。
上述胜肽的功能等價(functional equivalent)為一條具有與上述胜肽胺基酸序列至少60%(例如85%、90%或95%)相同的胺基酸序列的胜肽且該胜肽具有與上述胜肽相同的胜肽活性稱之。
可利用文獻(Karlin and AltschulProc. Natl. Acad. Sci.
USA 87:2264-68,1990,as modified in Karlin and AltschulProc. Natl. Acad. Sci.
USA 90:5873-77,1993)所提供的演算法認定兩胺基酸序列的相似性(percent identity)。於BLASTN與BLASTX程式(2.0版)(Altschul et al.J. Mol. Biol.
215:403-10,1990)中納入上述演算法。可依BLASTX程式(分數=50,字長=3)進行BLAST蛋白質搜尋,以獲得與本發明蛋白質分子一致的胺基酸序列。兩序列間存在的空隙(gaps),可使用如文獻(Altschul et al.,Nucleic Acids Res.
25:3389-3402,1997)所述考慮空隙的BLAST(gapped BLAST)。當使用BLAST與考慮空隙的BLAST程式時,可使用各別程式(亦即BLASTX與BLASTN)的預設參數(default parameters)。
可藉由已知的化學合成法(Creighton,Proteins;Structures and Molecular Principles,W. H. Freeman and Co.,NY,1983)製備上述胜肽。上述胜肽合成法的典型範例可包括,但不限定於,液相或固相合成、片段縮合(fragment condensation)以及F-MOC或T-BOC化學法(Chemical Approaches to the Synthesis of Peptides and Proteins,Williams et al.,Eds.,CRC Press,Boca Raton Fla.,1997;and A Practical Approach,Atherton & Sheppard,Eds.,IRL Press,Oxford,England,1989)。
此外,可藉由基因工程技術製備上述胜肽。首先,根據傳統方法建構編碼上述胜肽的一DNA序列。可利用適當引子(primers)藉由聚合酶鏈反應(polymerase chain reaction,PCR)放大建構DNA序列。亦可選擇性地藉由一已知的標準裝置,例如自動化DNA合成儀(Biosearch或Applied Biosystems)合成DNA序列。之後,將建構的DNA序列插入一載體(vector),其包括一或多個與DNA序列相連的表現控制序列(expression control sequences)(例如啟動子(promoters)、加強子(enhancers)等)。值得注意的是,表現控制序列調節DNA序列的表現。之後,以最終的重組表現載體(recombinant expression vector)轉化一宿主細胞(host cell)。於一培養基與一適合誘導DNA序列表現的培養條件下,培養最終的轉化產物。之後,自細胞培養中重新獲得一藉由DNA序列所編碼大體純的胜肽。可藉由已知的傳統方法(例如色層分析法)重新獲得上述胜肽。此處所提及”大體純的胜肽”一詞係表示本發明胜肽大體上無任何其他來自宿主蛋白質的摻雜。本發明合成胜肽的基因工程方法可見於下列文獻:Maniatis et al.,Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Laboratory,1982,Sambrook et al.,Molecular Cloning: A-Laboratory Manual,Cold Spring Harbor Press,N.Y.,Second(1998) and Third (2000) Edition,Gene Expression Technology,Methods in Enzymology,Genetics and Molecular Biology,Methods,in Enzymology,Guthrie & Fink(eds.),Academic Press,San Diego,Calif.,1991,以及Hitzeman et al.,J. Biol. Chem.,255:12073-12080,1990。
本發明利用噬菌體胜肽呈現技術(phage peptide display technique)篩選對血腦障壁(BBB)具有特異性的胜肽。
本發明之一實施例,提供一傳輸系統,包括:一載體,具有一表面;一藥物或一染料,包覆於載體內;以及一經分離之胜肽,包括一胺基酸序列SEQ ID NO: 1,接枝於載體之表面。
上述載體可包括高分子奈米粒子、固態脂質奈米粒子、高分子微胞(micelle)、微脂體(liposome)、微乳化液(microemulsion)或脂質奈米粒子。在一實施例中,選擇微脂體作為載體。微脂體可包括一第一脂質、一第二脂質或膽固醇。第一脂質可為一磷脂質(phospholipid),例如磷脂醯膽鹼(phosphatidyl choline,PC)、大豆磷脂醯膽鹼(soy phosphatidyl choline,SPC)、氫化大豆磷脂醯膽鹼(hydrogenated soy phosphatidyl choline,HSPC)或磷脂醯乙醇胺(phosphatidyl ethanolamine,PE)。第二脂質可包括二棕櫚酸磷脂醯甘油(dipalmitoyl phosphatidyl glycerol,DPPG)或二硬脂酸磷脂醯乙醇胺-聚乙二醇(distearoylphosphatidylethanolamine-polyethyleneglycol,DSPE-PEG)。於微脂體組成中,第一脂質具有一莫耳分率大體介於60~70之間,第二脂質具有一莫耳分率大體介於1~10之間,膽固醇具有一莫耳分率大體介於20~30之間,均以載體為100莫耳分率。在另一實施例中,選擇高分子微胞作為載體。當兩性分子(amphiphiles)置於水中時,即形成微胞,其包含一可溶解親脂性物質由親脂性片段所組成的內部核心以及一作為親脂性核心與外在水性環境之間一穩定介面的外部親水性暈(corona)。由於高分子具有高度多樣性、生物相容性、生物可分解性並且提供多重官能基與試驗分子形成共軛,使得高分子微胞的使用已獲得重視。類似其低分子量類似物,兩性高分子在水中會組合排列形成高分子微胞,以暴露於水性環境下的親水性高分子鏈暈穩定其親脂性核心。對於生理環境中顯現低溶解度、非期望藥物動力學或低穩定度的化合物而言,高分子微胞可作為有效的載體。藉由維持微胞於一分散狀態以及藉由立體-穩定效應降低與細胞及蛋白質的非期望藥物作用,親水性外殼對高分子配方的藥物行為有很大的貢獻。高分子微胞的尺寸範圍自大約10nm至大約100nm,通常,其尺寸分布是窄的。在生物環境作用下,由於藥物受到良好保護避免了可能的失活(inactivation),因此,高分子微胞可增加藥物生物可利用性與藥物滯留時間。高分子微胞已廣泛地研究作為低水溶性藥物例如紫杉醇(paclitaxel)、消炎痛(indomethacin)、兩性黴素B(amphotericin B)、阿黴素(adriamycin)以及二氫睪固酮(dihydrotestosterone)的注射藥物配方的傳輸介質。整體而言,高分子微胞已證實為一非常有效的藥物傳輸工具。
上述藥物可包括合成藥物、胜肽藥物、蛋白質藥物或核酸藥物。核酸藥物可包括質體DNA(plasmid DNA)、反意義寡核酸(antisense oligonucleotide)或RNAi。
上述胜肽可包括一由12個胺基酸殘基所組成的胺基酸序列KYLAYPDSVHIW(SEQ ID NO: 1)。值得注意的是,上述胜肽於細胞外(in vitro
)與細胞內(in vivo
)皆可結合至血腦障壁(BBB)。
上述經分離之胜肽具有一莫耳分率大體介於0.1~5之間,以載體為100莫耳分率。
本發明提供一種新穎的胜肽配體(BP02,具有一胺基酸序列SEQ ID NO: 1 KYLAYPDSVHIW),可標靶並通過血腦障壁(BBB)。本發明接枝有上述胜肽配體包覆藥物的載體當進入腦部時可避免藥物遭受破壞。
【實施例1】
體外血腦障壁模式之建立
體外血腦障壁模式的建立是利用RBE4細胞與C6細胞進行共同培養(co-culture),將其分別培養在一膠原蛋白處理過之半通透薄膜(Transwell-COL薄膜,孔洞尺寸0.4μm,購自Corning Costar,美國)的兩面,來模擬生理血腦障壁(BBB)狀態。上述半通透薄膜形成於一Transwell-COL插入皿(insert)的底部,並將Transwell-COL插入皿插入至一12-井盤中的每一井。每一井藉由Transwell-COL插入皿區分為一頂區(apical compartment)與一基底區(basal compartment)。首先,將C6細胞(105
cells/filter)均勻地種至Transwell-COL薄膜的反面,並允許C6細胞沉降貼附至薄膜1小時。之後,加入C6細胞培養基(以15%馬血清(horse serum)、2.5%胎牛血清(fetal bovine serum,FBS)以及盤尼西林(penicillin)/鏈黴素(streptomycin)(分別為100U/ml與100μg/ml)所補充的Ham’F10)至Transwell-COL插入皿的內部(井的頂區)與外部(井的基底區)。培養2天後,移去C6細胞培養基。之後,加入一共同培養基(co-culture medium)(pH 7.2)(α-MEM: Ham’s F-10(1:1)混合培養基,包含10%胎牛血清(FBS)、bFGF(1ng/ml)以及盤尼西林(penicillin)/鏈黴素(streptomycin)(分別為100U/ml與100μg/ml))至井的基底區。接著,將RBE4細胞(105
cells/filter)種至Transwell-COL薄膜的正面(井的頂區內),並與C6細胞於37℃培養箱(5% CO2
)中進行培養6~7天。每兩天更新一次共同培養基。
【實施例2】
體外血腦障壁模式
滲透度之評估
在RBE4細胞與C6細胞共同培養6~7天後,利用一共同培養插入皿(co-cultured insert)驗證體外血腦障壁模式的緻密度。首先,將插入皿之共同培養基移除。共同培養插入皿以HBSS緩衝液洗滌3次,再於HBSS緩衝液中37℃溫浴30min。移去HBSS緩衝液後,加入1.5ml新鮮HBSS緩衝液至井的基底區,再將0.5ml FLU溶液(1μg/ml FLU於HBSS緩衝液中)加入井的頂區,於37℃進行傳輸4小時。收集每一基底區中的樣品溶液,並進行FLU螢光分析(λEX
和λEM
分別為460nm以及515nm)(Intelligent Fluorescence Detector,FP2020,JASCO)。最後,利用下列公式計算出FLU滲透係數P
(cm/min),以確保體外血腦障壁模式會形成緻密的細胞層(P
<8×104
cm/min)。
【實施例3】
BP02胜肽之篩選
利用複雜性為2.7×109
的商業化Ph.D.-12TM
噬菌體呈現胜肽庫(phage-displayed peptide library)針對RBE4細胞進行淘選(panning)並以體外血腦障壁(BBB)模式進行穿透篩選(transmigration screening)。首先,將RBE4細胞種於6-井中,並於37℃培養直至細胞聚滿(confluence)。以PBS緩衝液洗滌聚滿的RBE4細胞層3次,並以BSA溶液(5mg/ml於PBS緩衝液中)先阻絕噬菌體與細胞的非專一結合(37℃,10分鐘)。將胜肽庫的噬菌體(1.5x1011
pfu)導入細胞,於4℃歷時2小時。以0.1% TBST緩衝液(50mM Tris-鹽酸,pH 7.4,150mM氯化鈉,0.1% v/v Tween-20)洗滌細胞6次,並於4℃藉由剝除洗液(0.2M甘胺酸-鹽酸,pH 2.2)將附著的噬菌體洗掉。洗出液立即以150μl 1M Tris-鹽酸緩衝液(pH 9.0)進行中和(第一次淘選)。將洗出液的噬菌體如他處所述進行放大與純化。利用第一次淘選的放大噬菌體(1.5x1011
pfu)進行第二回合篩選(第二次淘選,除將0.1% TBST緩衝液更換為0.3% TBST緩衝液,其餘均如第一次淘選所述)。以第二次淘選的放大噬菌體開始進行通過體外血腦障壁模式的噬菌體篩選(phage screening)。體外血腦障壁模式的建立如前所述。以HBSS緩衝液洗滌井並於HBSS緩衝液中溫浴30min後,將第二次淘選的放大噬菌體(7.0x1010
pfu)導入頂區,於37℃歷時4小時。收集基底區的樣品溶液並將溶液中的穿透噬菌體進行放大與純化(第一次transwell篩選)。以前次transwell篩選基底區的放大噬菌體(7.0x1010
pfu)開始進行第二至第三回合的transwell篩選。將第三次transwell篩選的放大噬菌體導入RBE4細胞進行另外三次的連續淘選。將最後一次淘選的篩選噬菌體進行放大與定序。一條新穎的胜肽(BP02,具有一胺基酸序列SEQ ID NO: 1)即完成篩選確認。
【實施例4】
BP02噬菌體之穿透率試驗
BP02噬菌體的穿透率試驗在RBE4細胞與C6細胞共同培養6~7天後進行。首先,將共同培養transwell的共同培養基移除。之後,每一井以HBSS緩衝液洗滌3次,再於HBSS緩衝液中37℃溫浴30min。移去HBSS緩衝液後,加入新鮮的HBSS緩衝液至井的基底區。將野生型(wild type)噬菌體以及BP02噬菌體(約1011
pfu/insert)分別加入井的頂區,於37℃進行傳輸1小時。之後,收集基底區中20μl的樣品溶液,並利用10倍序列稀釋的方式進行噬菌體滴定(phage titering),以計算出噬菌體的穿透率。結果如第1圖所示。BP02噬菌體的穿透率為控制組(野生型噬菌體)的14.8倍。
【實施例5】
BP02噬菌體之RBE4細胞結合試驗(藉由OPD呈色劑受質法(OPD chromophore substrate method))
將一96-井盤以膠原蛋白I(collagen I)進行塗層處理(37℃,1小時)。然後,再將RBE4細胞(104
細胞/井)培養於膠原蛋白處理過的96-井盤中。兩天後,將細胞用PBS緩衝液洗滌2次,以完全去除胎牛血清(FBS)。分別加入野生型(wild type)噬菌體以及BP02噬菌體至細胞,於37℃作用1小時。細胞經PBS緩衝液洗滌3次後,利用3%甲醛(formaldehyde)進行細胞固定(室溫,10分鐘)。細胞經PBS緩衝液洗滌4次後,加入HRP-conjugated anti-M13 mAb(1:1000)至細胞,於37℃作用1小時。細胞經PBS緩衝液洗滌4次後,加入一OPD呈色劑(chromophore)溶液至細胞,於37℃反應,直至呈現棕色為止(約2小時)。利用ELISA reader(波長490nm)測量最終溶液的OD值。結果如第2圖所示。結果顯示BP02噬菌體對RBE4細胞的結合以劑量依賴(dose-dependent)的方式發生。此外,控制組(野生型噬菌體)並未發現或僅有少許反應。
【實施例6】
BP02噬菌體之RBE4細胞結合試驗(藉由螢光劑酪胺受質法(fluorophore tyramide substrate method))
將蓋玻片置於12-井盤中並以膠原蛋白I(collagen I)進行塗層處理(37℃,1小時)。然後,再將RBE4細胞(1×105
細胞/井)培養於該12-井盤中。一天後,將細胞用PBS緩衝液洗滌2次,以完全去除胎牛血清(FBS)。之後,利用4%甲醛(formaldehyde)進行細胞固定(室溫,10分鐘)。細胞經PBS緩衝液洗滌2次後,加入1%雙氧水至細胞,以降低內生性活性(endogenous activity)(室溫,10分鐘)。細胞經PBS緩衝液洗滌2次後,加入1% BSA緩衝液至細胞以阻絕噬菌體與細胞的非專一結合(室溫,30分鐘)。之後,分別加入野生型(wild type)噬菌體以及BP02噬菌體(1012
pfu/well)至細胞,於室溫作用1小時。細胞經PBS緩衝液洗滌3次後,加入HRP-conjugated anti-M13 mAb(1:5000)至細胞,於室溫作用1小時。細胞經PBS緩衝液洗滌3次後,加入一螢光劑酪胺(fluorophore tyramide)溶液至細胞,於室溫反應10分鐘。細胞經PBS緩衝液洗滌3次後,將細胞固定並利用螢光顯微鏡進行照相。影像顯示BP02噬菌體對RBE4細胞的結合性非常強。控制組(野生型噬菌體)無法結合至RBE4細胞。
【實施例7】
標記rhodamine-PE之BP02微脂體之製備
(1) 標記rhodamine-PE之微脂體之製備
混合70mol%大豆磷脂醯膽鹼(soy phosphatidyl choline,SPC)、30mol%膽固醇、2mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇(distearoylphosphatidylethanolamine-polyethyleneglycol,DSPE-PEG2000)、1mol% rhodamine-PE以及3ml氯仿於一圓底燒瓶。以一旋轉濃縮機移除氯仿後,形成一脂質薄膜產物於燒瓶中。之後,加入PBS緩衝液以使脂質薄膜形成水合物,並以超音波震盪分散水合物。於60℃,以一擠壓機對分散產物依序使用200nm、100nm與50nm過濾器進行特定尺寸的標記rhodamine-PE微脂體製備。對於每一過濾器,分別進行擠壓製程10次。微脂體的顆粒尺寸(PS)為100±20nm。
(2) DSPE-PEG2000-BP02之製備
將3.2mg半胱氨酸(cysteine)-BP02溶解於10mM磷酸緩衝液(pH 6.5),以形成一半胱氨酸-BP02溶液(2mM)。將5.9mg二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺(distearoylphosphatidylethanolamine-polyethyleneglycolmaleimide,DSPE-PEG-maleimide)溶解於10mM磷酸緩衝液(pH 6.5),以形成一二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺溶液(2mM)。於4℃,將二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺溶液緩慢加入半胱氨酸-BP02溶液(莫耳比=1:1)並攪拌4小時,以製備二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG2000-BP02)。以5,5'-二硫代雙-(2-硝基苯甲酸)(5,5'-dithiobis-(2-nitrobenzoic acid),DTNB)(Ellman's試劑)計算,其接枝率為61%。
(3) 標記rhodamine-PE之BP02微脂體與標記rhodamine-PE之PEG微脂體之製備
將上述(2)中的3mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG2000-BP02)溶液與3mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇(DSPE-PEG2000)溶液分別加入上述(1)中的標記rhodamine-PE微脂體溶液進行後插入(post-insertion),於60℃水浴下進行1小時,以製備標記rhodamine-PE的BP02微脂體與標記rhodamine-PE的PEG微脂體。
【實施例8】
標記rhodamine-PE之BP02微脂體之RBE4細胞結合試驗
將蓋玻片置於12-井盤中並以膠原蛋白I(collagen I)進行塗層處理(37℃,1小時)。然後,再將RBE4細胞(1×105
細胞/井)培養於該12-井盤中。一天後,將細胞用PBS緩衝液洗滌2次。之後,分別加入標記rhodamine-PE的BP02微脂體與標記rhodamine-PE的PEG微脂體(2μg/ml rhodamine-PE)至細胞,於37℃作用2小時。細胞經PBS緩衝液洗滌3次後,以一螢光顯微鏡拍攝影像。
根據螢光照片,經標記rhodamine-PE的BP02微脂體處理的RBE4細胞較經標記rhodamine-PE的PEG微脂體處理的RBE4細胞具有較強螢光。也就是說,標記rhodamine-PE的BP02微脂體對RBE4細胞產生強的結合能力並藉由BP02配體迅速為RBE4細胞所吞噬。
【實施例9】
包覆FLU之BP02微脂體之製備
混合70mol%大豆磷脂醯膽鹼(SPC)、30mol%膽固醇以及3ml氯仿於一圓底燒瓶。以一旋轉濃縮機移除氯仿後,形成一脂質薄膜產物於燒瓶中。之後,加入50mM FLU溶液(FLU於PBS緩衝液中)以使脂質薄膜形成水合物,並以超音波震盪分散水合物。於60℃,以一擠壓機對分散產物依序使用200nm、100nm與50nm過濾器進行特定尺寸之包覆FLU的微脂體製備。對於每一過濾器,分別進行擠壓製程10次。利用凝膠過濾法移除未包覆的FLU染料以純化包覆FLU的微脂體。微脂體的顆粒尺寸(PS)為100±20nm。此外,將3mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG2000-BP02)溶液與3mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇(DSPE-PEG2000)溶液分別加入微脂體溶液進行後插入(post-insertion),於60℃水浴下進行1小時,以製備包覆FLU的BP02微脂體與包覆FLU的PEG微脂體,並保存於透析袋(MWCO=1000Da)中移除殘留的FLU。
【實施例10】
包覆FLU之BP02微脂體之穿透率試驗
包覆FLU的BP02微脂體的穿透率試驗在RBE4細胞與C6細胞共同培養6~7天後進行。首先,將共同培養transwell的共同培養基移出。之後,每一井以HBSS緩衝液洗滌3次,再於HBSS緩衝液中37℃溫浴30min。移去HBSS緩衝液後,加入新鮮的HBSS緩衝液至井的基底區。之後,將包覆FLU的BP02微脂體與包覆FLU的PEG微脂體(約150ng/ml)分別加入井的頂區,於37℃進行傳輸1小時。之後,收集基底區中各別的樣品溶液,並以甲醇稀釋10倍。以甲醇打破微脂體後,進行FLU螢光分析(λEX
和λEM
分別為460nm以及515nm)(Intelligent Fluorescence Detector,FP2020,JASCO),並計算穿透率。包覆FLU的BP02微脂體的穿透率為包覆FLU的PEG微脂體的3.3倍,表示BP02配體有助微脂體通過血腦障壁(BBB),如第3圖所示。
【實施例11】
包覆Cy5.5之BP02微脂體之製備
於一25ml減壓濃縮瓶中,於60℃水浴下,將41.7mg大豆磷脂醯膽鹼(SPC)、4.2mg氫化大豆磷脂醯膽鹼(hydrogenated soy phosphatidyl choline,HSPC)、4.1mg二棕櫚酸磷脂醯甘油(dipalmitoyl phosphatidyl glycerol,DPPG)、2.1mg膽固醇、7.8mg Brij 76、15.4mg二硬脂酸磷脂醯乙醇胺-聚乙二醇(DSPE-PEG2000)以及5mg Cy5.5染料溶解於5ml乙醇,以形成一混合溶液。對混合溶液進行減壓濃縮後,加入5ml蔗糖溶液(10%)至混合溶液,以形成一水合物。之後,於4℃,以超音波震盪分散水合物。水合物經過濾後(分別使用0.45μm與0.2μm過濾器),形成包覆Cy5.5的PEG微脂體(CYL003)。此外,將二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG2000-BP02)(0.178mg)溶液加入CYL003,於60℃水浴下反應1小時,以製備包覆Cy5.5的BP02微脂體(BP02-3.6%)。
【實施例12】
包覆Cy5.5之BP02微脂體之生物分佈試驗
將一近紅外光螢光染料-Cy5.5磷醯亞胺(phosphoramidite)(購自GE Healthcare Life Sciences)包覆於BP02微脂體或PEG微脂體作為一顯示生物分佈與腦中螢光強度的光學試劑。以5mg/kg Cy5.5微脂體對老鼠(4~6週BALB/c裸鼠)進行尾靜脈注射,藉由吸入性麻醉劑isoflurane進行麻醉,並於注射後10、20、30、60、120、180與240分鐘擷取影像。藉由IVIS200系統(Xenogen Corp.)利用一秒鐘曝光時間(f/stop=8,Binning=8)擷取影像,並利用活體影像與IGOR軟體圈選全腦作為感興趣區域(regions of interest,ROI)進行分析。於注射後10分鐘利用每隻動物的起始螢光值對螢光強度進行正規化並將其定義為每球面度(steradian)每平方公分每秒的光子數(p/s/cm2
/sr)。結果如第4圖所示。結果顯示BP02配體有助微脂體累積於老鼠腦中。
【實施例13】
包覆腦內啡-1(endomorphin-1)之BP02微脂體之製備
關於止痛的研究,利用一大豆磷脂醯膽鹼(SPC)、氫化大豆磷脂醯膽鹼(HSPC)、二棕櫚酸磷脂醯甘油(DPPG)、膽固醇、Brij76與维生素E聚乙二醇1000琥珀酸酯(d-alpha-tocopheryl polyethylene glycol 1000 succinate,TPGS)莫耳比為20:2:2:2:4:1的混合物包覆腦內啡-1(endomorphin-1,EM-1)。將所有材料置於一12.5ml ZrO2研缽(mortar),並置入5顆ZrO2珠(直徑10mm),以開始500rpm,1小時的研磨製程(使用機台為Planetary Micro Mill,Pulverisette 7)。之後,加入10mM PBS(pH 7.4)並於室溫下攪拌1小時以使該膏狀研磨物形成一水合物,以製備包覆腦內啡-1(EM-1)的微脂體。此外,將1.5mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG2000-BP02)溶液與1.5mol%二硬脂酸磷脂醯乙醇胺-聚乙二醇(DSPE-PEG2000)溶液分別加入微脂體溶液進行後插入(post-insertion),於4℃攪拌隔夜,以製備包覆腦內啡-1(EM-1)的BP02微脂體(NL563-BP02)與包覆腦內啡-1(EM-1)的PEG微脂體(NL563)。
【實施例14】
熱板試驗
為驗證腦部標靶(brain-targeting),利用熱板試驗(hot-plate test)的動物模式評估BP02配體的止痛效果。熱板試驗係藉由讓老鼠腳墊與熱板產生接觸以評估熱引起的疼痛反射作用。動物對熱板的反應反映出痛覺行為(nociceptive behavior),其包含中樞神經系統中複雜的神經網路。
主要來說,將老鼠各別置於55℃熱板裝置上,並量測開始舔後肢的時間。基本潛伏期(basal latency)大約2秒,無反應的終止時間(T2)設定為45秒。舔後肢反應的抑制以最大可能效果百分比(% MPE)表示之,以下式計算[(T1-T0)/(T2-T0)]×100,其中T0與T1為靜脈注射腦內啡-1(EM-1)配方前後的熱板潛伏期(hot-plate latency)。於注射配方(7.5mg/kg腦內啡-1(EM-1))後10、20、30、60、120、180與210分鐘測定熱板潛伏期,並計算MPE%對時間(分鐘)圖的AUC(曲線下面積)值。
單獨給予腦內啡-1(EM-1) 7.5mg/kg劑量並未降低老鼠的痛覺反應(未顯示數據)。相同劑量的NL563配方僅於給藥後30分鐘有少許止痛效果。然而,NL563-BP02配方明顯產生止痛作用,於給藥後120分鐘達到熱板潛伏期提升約25%,並延長其止痛作用3小時以上。根據AUC值,NL563-BP02的效果比NL563高4倍,並於給藥後120分鐘與180分鐘在配方間具有統計上的明顯差異(P
<0.05,單尾T檢定),此即表示標靶配方(NL 563-BP02)在循環過程中可避免藥物降解,並有助傳輸藥物至腦神經系統,如第5、6圖所示。
【實施例15】
標記rhodamine-PE之BP02高分子微胞之製備
(1) DSPE-PEG5000-BP02之製備
將1mg半胱氨酸(cysteine)-BP02溶解於10mM磷酸緩衝液(pH 6.5),以形成一半胱氨酸-BP02溶液(0.625mM)。將3.7mg二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺(distearoylphosphatidylethanolamine-polyethyleneglycol maleimide,DSPE-PEG5000-maleimide)溶解於10mM磷酸緩衝液(pH 6.5),以形成一二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺溶液(0.625mM)。於4℃,將二硬脂酸磷脂醯乙醇胺-聚乙二醇-馬來醯亞胺溶液緩慢加入半胱氨酸-BP02溶液(莫耳比=1:1)並攪拌隔夜,以製備二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG5000-BP02)。以5,5'-二硫代雙-(2-硝基苯甲酸)(5,5'-dithiobis-(2-nitrobenzoic acid),DTNB)(Ellman's試劑)量測,其接枝率為81%。
(2) 標記rhodamine-PE之BP02高分子微胞之製備
將10mg 2-乙基-2-噁唑啉-聚乳酸(PEOZ-PLA)共聚物(MW=15,000)與0.05mg rhodamine-PE(Rh-PE)混合並溶解於1ml氯仿。移除氯仿,加入1ml 10mM PBS(pH 7.4)以使得到的薄膜再次形成水合物,並進行超音波震盪(XL2020,Misonix Inc.,美國)1分鐘,以獲得標記rhodamine-PE的高分子微胞(控制組)。另將5%莫耳比的二硬脂酸磷脂醯乙醇胺-聚乙二醇-BP02(DSPE-PEG5000-BP02)插入微胞,如BP02高分子微胞(polymeric micelles)。以雷射粒徑分析儀(laser particle size analyzer)(N4 plus,Coulter Electronics,美國)量測,控制組與BP02配方的平均顆粒尺寸分別為57.3nm與66.9nm。
【實施例16】
體外RBE4細胞對BP02高分子微胞之結合/攝入
為驗證BP02胜肽位於其他奈米顆粒上的標靶能力,即配製耦接BP02胜肽並標記rhodamine的高分子微胞(PMs)進行細胞結合(binding)/攝入(uptake)研究。首先,將RBE4細胞種於膠原蛋白(collagen I)處理過的蓋玻片上,並於37℃培養隔夜。將控制組或BP02高分子微胞導入RBE4細胞,並於37℃無血清培養基中培養1小時。培養後,以PBS洗滌細胞3次,將細胞固定並利用螢光顯微鏡進行照相。對於以BP02高分子微胞處理過的細胞,其細胞螢光強度(cell-associated fluorescence)明顯比控制組高,此即表示其對RBE4細胞具有較高結合力。
【實施例17】
R
BE4細胞對BP02高分子微胞之結合/攝入量
對標記rhodamine高分子微胞的細胞結合/攝入進行定量。主要來說,將RBE4細胞以2×105
細胞/井的密度種於膠原蛋白(collagen I)處理過的12-井盤中,並於37℃培養2天。加入Rh-PE濃度為1.25μg/ml的控制組或BP02高分子微胞,並於37℃無血清培養基中培養1小時。移除培養基後以PBS洗滌細胞3次。之後,加入細胞溶解緩衝液(cell lysis buffer)(ProteoJETTM
Mammlian Cell Lysis reagent,K0301,Fermentas)溶解細胞,並以一螢光光譜儀(fluorescence spectrophotometer)(Intelligent Fluorescence Detector,FP2020,JASCO)(激發波長568nm,放射波長590nm)量測細胞溶解物(cell lysates)中的Rh-PE濃度。另一方面,收集一井中的細胞並計算細胞數目。細胞的結合/攝入以每104
細胞含多少毫微克表示之。當與控制組高分子微胞相比時,對於以BP02高分子微胞處理過的細胞,其細胞螢光明顯增加(P<0.00005,雙尾T檢定),結果如第7圖所示。
雖然本發明已以較佳實施例揭露如上,然其並非用以限定本發明,任何熟習此項技藝者,在不脫離本發明之精神和範圍內,當可作更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
<110> 財團法人工業技術研究院
<120> 經分離之胜肽及包含此胜肽之傳輸系統
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 人工合成胜肽序列
<400> 1
第1圖係根據本發明之一實施例,顯示利用一transwell系統評估野生型(wild type)與BP02噬菌體通過體外血腦障璧的能力;
第2圖係根據本發明之一實施例,顯示野生型(wild type)與BP02噬菌體對RBE4細胞的結合程度;
第3圖係根據本發明之一實施例,顯示以BP02胜肽修飾包覆FLU的微脂體的穿透率;
第4圖係根據本發明之一實施例,顯示包覆Cy5.5的BP02微脂體在老鼠的腦部累積情形;
第5圖係根據本發明之一實施例,顯示PEG微脂體(NL563)與BP02微脂體(NL563-BP02)其熱板試驗之MPE(%)對時間(分鐘)的關係;
第6圖係根據本發明之一實施例,顯示PEG微脂體(NL563)與BP02微脂體(NL563-BP02)其熱板試驗的AUC值;
第7圖係根據本發明之一實施例,顯示BP02高分子微胞結合/攝入RBE4細胞的定量結果。
Claims (14)
- 一種經分離之胜肽,由SEQ ID NO:1胺基酸序列所組成。
- 一種傳輸系統,包括:一載體,具有一表面;一藥物或一染料,包覆於該載體內;以及一如申請專利範圍第1項所述之經分離之胜肽,接枝於該載體之該表面。
- 如申請專利範圍第2項所述之傳輸系統,其中該載體包括高分子奈米粒子、固態脂質奈米粒子(solid lipid nanoparticle)、高分子微胞(micelle)、微脂體(liposome)、微乳化液(microemulsion)或脂質奈米粒子(lipid-based nanoparticle)。
- 如申請專利範圍第3項所述之傳輸系統,其中該微脂體包括一第一脂質、一第二脂質及膽固醇。
- 如申請專利範圍第4項所述之傳輸系統,其中該第一脂質為一磷脂質(phospholipid)。
- 如申請專利範圍第5項所述之傳輸系統,其中該磷脂質包括磷脂醯膽鹼(phosphatidyl choline,PC)、大豆磷脂醯膽鹼(soy phosphatidyl choline,SPC)、氫化大豆磷脂醯膽鹼(hydrogenated soy phosphatidyl choline,HSPC)或磷脂醯乙醇胺(phosphatidyl ethanolamine,PE)。
- 如申請專利範圍第4項所述之傳輸系統,其中該第二脂質包括二棕櫚酸磷脂醯甘油(dipalmitoyl phosphatidyl glycerol,DPPG)或二硬脂酸磷脂醯乙醇胺-聚乙二醇(distearoylphosphatidylethanolamine-polyethyleneglycol,DSPE-PEG)。
- 如申請專利範圍第4項所述之傳輸系統,其中該第一脂質具有一莫耳分率大體介於60~70之間,該第二脂質具有一莫耳分率大體介於1~10之間,該膽固醇具有一莫耳分率大體介於20~30之間,以該載體為100莫耳分率。
- 如申請專利範圍第2項所述之傳輸系統,其中該藥物包括合成藥物、胜肽藥物、蛋白質藥物或核酸藥物。
- 如申請專利範圍第9項所述之傳輸系統,其中該核酸藥物包括質體DNA(plasmid DNA)、反意義寡核酸(antisense oligonucleotide)或RNAi。
- 如申請專利範圍第2項所述之傳輸系統,其中該經分離之胜肽具有一莫耳分率大體介於0.1~5之間,以該載體為100莫耳分率。
- 如申請專利範圍第2項所述之傳輸系統,其中該經分離之胜肽具有一莫耳分率大體介於0.1~2.5之間,以該載體為100莫耳分率。
- 一種經分離之胜肽,由與SEQ ID NO:1具有至少90%相同度之胺基酸序列所組成。
- 一種經分離之胜肽,由與SEQ ID NO:1具有至少95%相同度之胺基酸序列所組成。
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