TWI392509B - 以紅血球微囊作為奈米藥物遞送系統 - Google Patents
以紅血球微囊作為奈米藥物遞送系統 Download PDFInfo
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Description
本發明係關於一種奈米粒子,尤指一種用於奈米藥物遞送系統。
奈米粒子在藥物遞送上的定義指的是小於1微米(<1 μm)之膠體粒子,該定義包括藉由基質將藥物吸附、溶解或分散於其中之單片奈米粒子(奈米球),以及其中藥物係被限制在由殼狀壁面包圍之水性或油性核心中的奈米膠囊。或者,藥物可經共價鍵結在表面或基質內。奈米粒子是由生物相容性及生物可降解物質等所組成,像是天然的(例如:動物膠、白蛋白)或合成的(例如:聚交酯、聚烷基氰基丙烯酸酯(polyalkylcyanoacrylates)聚合物或固態脂質。在人體內部,裝載於奈米粒子中之藥物通常係藉由擴散、膨脹、浸蝕或降解等作用,而從基質釋放出(Gelperina等人(2005)“奈米粒子藥物遞送系統於結核病化學療法之可能優勢”美國呼吸與危急照護醫學期刊Vol 172,1487-1490)。
當奈米粒子的獨特特徵(像是小尺寸及較大的表面積-對-質量比與物化性質等),在生物醫學應用上提供令人振奮之承諾的同時,也令人擔憂其潛在的毒性。例如在幹細胞追蹤中,超順磁氧化鐵(SPIO)奈米粒子已被公認可用於細胞磁共振造
影(MRI),做為一種細胞內標定細胞的工具,其在成功的幹細胞治療發展上扮演一重要的角色。因為原始超順磁性氧化鐵奈米粒子(native SPIO nanoparticles)的細胞內在化效率低,故曾經報導過數種對於SIPO奈米粒子之修正,以改善SIPO奈米粒子的細胞內化。但必須考量這些修正對於幹細胞可能造成的危險性。奈米粒子是經人為製造且是異種異體的事實,為奈米醫學應用上可能存在危險性的永久議題。
隨著囊泡形成並生成奈米大小的紅血球微囊(RDV),該血紅蛋白和薄膜所組成的血紅素也逐漸減少。紅血球微囊(RDV)和老化的紅血球可能被認為可從循環(circulation)系統中移除。在體內的滲透和氧化壓力下能夠使紅血球產生相似的囊泡,由於生物相容性及生物降解性原由,紅血球能攜帶不同生物活性基質的潛在可能性已經廣泛地被利用。使奈米粒子在生理狀態下安定,提供進行表面進一步反應的能力,及避免網狀內皮系統的吸收,藥物標的為藥物遞送的現代技術之一。
紅血球的直徑約為7.5-8 μm,比一般奈米粒子還大。除了巨噬細胞以外只有很少數細胞能夠吞噬這樣大的粒子。故產生含有且封裝物質之亞微米RDV,可消除這些問題並滿足對於奈米藥物遞送系統的需求。
本發明的目的之一係關於一種經分離的紅血球微囊,其中
含有經封裝之外來物質(encapsulated exogenous substance)。
於本發明之一項具體實施例,該經分離的紅血球微囊具有直徑不超過500、400、350或300 nm,被封裝之外來物質可包括至少一種選自螢光基團、核酸、超順磁化合物和治療試劑之物質。該分離的紅血球微囊能進入巨噬細胞以外之細胞中,而不需在其表面膜上進行任何修飾。
本發明的另一目的係關於一種將物質遞送進入細胞中之方法,其包括下列步驟:(a)將細胞與含有經封裝之外來物質的經分離紅血球微囊接觸;(b)讓細胞將該紅血球微囊內在化以得到已內在化紅血球微囊之細胞,藉此而將該物質遞送進入細胞中。
於本發明方法之另一項體實施例,其包括將細胞與具有直徑不超過500、400、350或300 nm之經分離的紅血球微囊接觸之步驟。
於本發明之另一具體實施例,該方法包括將細胞與含有至少一種選自螢光基團、核酸、胜肽、超順磁化合物和治療試劑之物質的經分離紅血球微囊接觸之步驟。該細胞為至少一種選自初級細胞、癌症細胞和幹細胞者。於本發明之一具體實施例,該細胞和其中所包含之紅血球微囊為自體移植的。
於本發明之另一具體實施例,該方法包括本發明包括將幹細胞與封裝有超順磁化合物的分離紅血球微囊接觸之步驟。本發明之方法可進一步包括藉由MRI來追蹤幹細胞內所含之超
順磁化合物的步驟。
於本發明之另一具體實施例,該方法包括將已內在化RDV之幹細胞投藥予患者的步驟,其中該幹細胞和RDV係從患者自體移植的。該方法可進一步包括利用MRI於活體內追蹤存在幹細胞的超順磁化合物,該超順磁化合物包括Fe3O4。
本發明的又另一目的係關於一種製造經分離的紅血球微囊(RDV)之方法,其包括下列步驟:(a)從血液樣本中製備出紅血球(RBC);(b)準備1 M CaCl2、390 mM EDTA和雙去離子水(ddH2O);(C)根據下述體積比例來混合RBCs、CaCl2、EDTA與ddH2O以得到含有RDV的混合物:(i)CaCl2:EDTA=1:1;(ii)RBC的體積為CaCl2之2.5~5倍;(iii)ddH2O的體積為RBC和CaCl2:EDTA的總合之差值。
於本發明之一項具體實施例,上述步驟(C)係在50℃以下完成。於本發明之另一具體實施例,前述之方法進一步包括將含有RDV混合物離心,以收集得該分離的RDV。
這些方法及其他目的將隨著下述的描述及圖示以揭示本創作之較佳實施例,本文於此所揭示的實施例於所有觀點,應被視為用以說明本創作,而非用以限制本創作。
在本發明所使用的特殊術語有其原本的意義,如下所用的某些特殊術語是提供熟悉該技藝者能更進一步了解本發明內
容。為了方便起見一些特殊術語將會使用斜體字或引號標示出來,但這些被標示出來的部分並不會影響到特殊術語本身的範圍或意義,就如同在本文中未被標示的文字一樣,也就是說同樣的事情會有一個以上的說法。本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明,而該實施範例僅作為輔助說明,並非用於限制本發明之範圍。
除非另有規定,本發明所涉及的科學和技術所用詞彙和一般普通技能所使用的詞彙為相同的,若是有所衝突的情況下,本發明將會給予名詞新的定義。
本發明所使用的”大約”意指的是所給予數值上下百分之十,最好是百分之十,或更進一步而言最好是百分之五的範圍,在此所給予數值數量為大約的意思,並沒有明文強制規定。
本發明所使用的”異種(xenogeneic)”指的是從不同物種的有機體中衍生或得到。
本發明所使用的”外生(exogenous)”指的是從外部所得到的原始物。
本發明所使用的”自體移植(autologous)”指的是來自相同的有機體。
本發明所使用的”初級細胞(primary cells)”指的是直接取自活的有機體(活體組織材料)之細胞。
本發明係關於一種紅血球微囊(RDV)及其應用作為藥物遞送系統。RDV呈現之特性包括能夠被細胞吸收,以及可做
為用於幹細胞細胞內標定與MRI之超順磁性氧化鐵(SPIO)的奈米載體。此外,自體移植之紅血球微囊(RDV)的優良生物相容性,可解決生物醫學上與奈米粒子應用相關的奈米毒物學議題。
根據本發明所呈現的各種實施例,下述各種儀器、裝置、方法和其相關結果者,實施例中為了方便讀者閱讀所使用的標題或副標題,並不被限制在本發明的範圍之內。此外,在此所提出和披露的某些理論,但無論他們是對還是錯,只要該創作是根據本發明所實施的,而不需考慮任何特定的理論或行動的計畫,都應被限制在本發明的範圍之內。
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明,而該實施範例僅作為輔助說明,並非用於限制本發明之範圍。
利用Ca2+-EDTA(Leonards,K.S.與S.Ohki(1983)“從人類及兔子的紅血球分離及鑑定出大的無細胞骨架囊泡”Biochim Biophys Acta 728(3):383-93),製備紅血球微囊(RDV)。
簡單地說,從5位男性及5位女性的健康捐贈者中得到30 ml的靜脈血液樣本,並和50 U/ml的肝磷脂(heparin)混合。血液樣品在1,700×g、4℃的條件下離心10分鐘後,把血漿
移除。將200微升的紅血球(大約2-2.4×106 RBCs)和各種比例的1 M CaCl2及390 mM EDTA在45℃的條件下混合30分鐘,以產生紅血球微囊(如表一所示)。將所有處理過的紅血球都置於相位差顯微鏡(phase contrast microscope,Olympus)下進行觀察。將該混合物在1,700×g、4℃的條件下離心10分鐘,離心後將上清液收集,接著再於1,600×g、4℃的條件下離心10分鐘,而得到超小囊泡(大約200 nm),在離心後將沈澱物以PBS清洗兩次,並再懸浮於PBS溶液中。
利用鐵超晶格套組(Iron-SL kit,Diagnostic化學品股份有限公司),根據製造商的說明,來分析紅血球微囊(RDV)中鐵離子的濃度。鐵超晶格(Iron-SL kit)中的色素原Ferene®會和二價
鐵反應,形成在595 nm吸收光的藍色色團,利用ELISA讀取機在595 nm下量測吸光度(OD)而收集數據。
利用鐳射光繞射粒徑分析儀(particle size analyzer)量測紅血球微囊(RDV)的大小(90 plus,Brookhaeven,儀器公司),而紅血球微囊(RDV)的直徑也是使用穿透式電子顯微鏡(TEM)來測量,將紅血球微囊(RDV)直接裝載於銅網(grid)(電子顯微鏡科學)上,額外的PBS被硝化纖維素膜所吸收。將所得到的裝載銅網在室溫下於乾燥箱中靜置過夜,再用TEM(Hitachi H-7650)進行觀察。
一般來說,將紅血球微囊(RDV)和10 μl含有欲被封裝物質之溶液,與100 μl的低滲透緩衝溶液(Na2HPO4/NaH2PO4,20 mM,pH 8),在4℃的條件下培育一個小時。將該混合物在1,600×g、4℃的條件下,離心10分鐘使微囊沈澱下來,而將其從含有非未經封裝物質之溶液中分離出。藉由將沈澱物再懸浮於PBS中,並以1,600×g於4℃下離心10分鐘來清洗微囊。在經第二次清洗後,將該囊泡於使用前最後一次再懸浮於PBS中。被封裝的物質如下表所示。
dsDNA產物(SEQ ID NO:3)利用前置引子,羧基螢光素(FAM)-T7(FAM-5’-TAATACGACTCACTATAGGG;SEQ ID NO:1;PURIGO BIOTECH,台灣)和反置引子,ferritin-3’Xho(GACTCGAGCTAGTCGTGCTTGAGAGTGAGG;SEQ ID NO:2;MDBio,台灣)進行PCR放大。該前置引子羧基螢光素(FAM)-T7為噬菌體T7啟動區DNA,反置引子ferritin-3’Xho為Xho I限制酵素序列以達成複製的目的。PCR複製條件如下:1×PCR緩衝溶液(50 mM KCl,10 mM Tris-HCl,pH 8.8,2.5 mM MgCl2,和0.1%,Triton X-100),dNTPs(0.05 mM),前置與反置引子(各0.1 μM),樣板DNA(1 ng/μl),與Taq DNA聚合脢(0.02 U/μl)。將最後的PCR產物以FAM標記(MDBio,台灣),關於FAM-ssDNAT7寡核苷酸,係由PURIGO BIOTECH(台灣)合成FAM-5’-TAATACGACTCACTATAGGG(SEQ ID NO:4)。關於
FAM-siRNA(陰性對照FAM),則係由MDBio(台灣)合成有義(sense):FAM-5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO:5),與反義(Anti-sense):5’-ACGUGACACGUUCGGAGAATT-3’(SEQ ID NO:6)。藉由MRI和流式細胞儀分析已封裝有物質的RDV。
其他經測試且能被封裝入RDV之物質包括:氧化鐵(T2對比)、釓(Gd,為用於MRI的T1對比試劑)、紫杉醇、鹽酸阿黴素(Doxorubicin hydrochloride)、原紫質環IX(用於光療法的一種光敏劑)、鹽酸5-氨基3-乙醯丙酸(鹽酸5-氨基3-乙醯丙酸的前藥)、1-B-D-5阿拉伯呋喃糖苷基胞嘧啶(Ara-c)以及全反式維生素甲酸(ATRA,Tretinoin,維他命A酸)。多肽包括胰島素、EPO、白細胞介素、EGF、賀爾蒙、細胞因子等,以及多醣類包括鏈脲黴素(Streptozotocin)、托吡酯(topiramate)、乳果糖(lactulose)、4-胍基-Neu5Acen等亦可被封裝入RDV中。每種被封裝物質各自擁有其特性,且被封裝的效率也會有所不同,因此很難推論出何種被封裝物具有最佳的封裝效率。
據發現,每種物質的裝載效率主要取決於他們本身的特性。顯然愈疏水性的藥物愈容易被封裝入RDV中。對於不同物質使用不同的封裝方法。可能有助於克服封裝困難性及增加封裝效率之因子包括,於低滲透壓的溶液中加入ATP和葡萄糖(Magnani,M.,L.Rossi,等人(1992)“對巨噬細胞靶定呈磷酸
化形式之抗病毒核苷類似物:活體外及活體內研究”Proc Natl Acad Sci USA 89(14):6477-81),所以顯然對於封裝所有物質而言並沒有一體適用的方法。
藉著將100 μg的Fe3O4(Taiwan Advanced Nanotech Inc.,Amino-TANBeads/USPIO-101)與10 μl的dd H2O混合,而製備得Fe3O4儲存溶液(100 μg/10 μl)。將10 μl儲存溶液和90 μl低滲透壓溶液及2 ml的DMEM混合來處理細胞,以測試單獨Fe3O4是否能進入細胞中。
Fe3O4封裝於RDV的製備方法如下:將RDV(100 μg溶於10 μl PBS)和10 μl的Fe3O4儲存溶液(100 μg/10 μl)及80 μl低滲透壓溶液混合,並在4℃下培育1小時。在處理細胞之前,將所得到Fe3O4封裝於RDV的混合物加入2 ml的DMEM中。
為了測試RDV是否能夠將Fe3O4遞送進入細胞中,遂將1.2×105 hMSCs以載體、RDV、Fe3O4、Fe3O4加RDV或Fe3O4被封裝於RDV,在37℃下處理1小時。將細胞以PBS清洗兩次,並利用胰蛋白酶化及於1,200×g離心5分鐘收取細胞,然後可利用1.5-T MRI系統進行MRI檢測。經過Fe3O4被封裝於RDV處理之細胞能夠被MR成像,證明RDV能夠攜帶,並將Fe3O4遞送進入幹細胞中。
NIH3T3細胞株(老鼠的胚胎纖維細胞)和B16細胞(小鼠黑色素瘤細胞株)生長在輔以10%胎牛血清中成長(FBS,Biological Industries)和1×青黴素-鏈黴素-兩性黴素B(Biological Industries)之DMEM培養液中。
人體骨髓間充質幹細胞(hMSCs)是從正常捐贈者的骨髓分離而來。簡單地說,將骨髓抽取液以1:1之比例(v/v)加到含有25 U/ml肝磷脂的低葡萄糖DMEM培養液中,再利用Ficoll-Paque密度梯度離心法(density gradient centrifugation)進行分離。收集富含hMSCs之低密度部分,以DMEM沖洗後,以每瓶5×107有核細胞存在於5 ml正規培養液的密度置入T25燒瓶中,該培養溶液是由低葡萄糖DMEM輔以10%胎牛血清(FBS;HyClone,Logan,UT)、4 mM左旋麸醯胺酸、100 U/ml的盤尼西林和100 μg/ml鏈黴素(Sigma-Aldrich)所組成。
在最初兩個星期之細胞貼壁和開始擴張期間,在第一週每星期加入兩次5 ml新鮮的生長培養基。每星期更換兩次培養基。當貼壁細胞達到60%~70%鋪滿時,以0.25%胰酶细胞消化液(trypsin-EDTA)將其脫離,並於1:3比例的重新放置到正規培養液中,進行連續繼代培養。所有培養物都保存在37℃,5% CO2和95%空氣的大氣中。
為了將RDV傳遞進入細胞內,遂將存在10 μl PBS中之
100 μg RDV和90 μl的低滲透壓溶液(即如前所述之低滲透壓溶解緩衝液)混合。將100 μl的混合物加到2 ml的DMEM中,而形成RDV的懸浮液製劑。將該RDV懸浮液加載於生長在玻片上的NIH3T3細胞上,並進行培育一段不同的時間。
B16或hMSCs細胞(1.2×105細胞)在2 ml的生長培養液(DMEM)中,生長於塗佈有poly-L-lysine的玻璃蓋玻片上,並以該RDV懸浮液處理一個小時。做為對照組,係將細胞與載劑(其係藉由將100 μl的低滲透壓溶液加入在2 ml的DMEM中而得)進行培育。
當載入RDV之後,將NIH3T3細胞於PBS中清洗並放置到PCR試管內,該PCR試管浸泡於水中並藉由MRI觀察RDV的細胞攝入。
在以封裝有Fe3O4之RDV處理過後,藉由離心將細胞沈澱在微量離心管底部。將該試管放置在水槽內,接著再將水槽放置到八頻道頭線圈(eight-channel head coil)中。使用1.5-T MR成像系統(Signa excite,GE Healthcare,USA)完成MRI。對於磁共振成像,係使用二維T2-加權快速自旋回波(Fast spin echo)再加上供應商(FSE-XL/90)所提供的序列(TR/TE ¼ 667/11.9 ms)。切片的厚度為1.4 mm(0.03 mm的差距),且視野(FOV)為16T8 cm。全部的掃描時間為3分鐘47秒且激發次數(NEX)
為6。接著由GE healthcare提供之工作站進行影像分析。
雄性balb/c小鼠(大約六個星期大)從國立台灣大學(台灣)動物中心所取得,根據動物實驗管理小組(IACUC)進行管理的步驟及規範。hMSCs經由載體或是封裝有Fe3O4之RDV處理過後,藉由胰蛋白酶消化收取並懸浮於PBS中。藉由在腹腔空間內射注入氯胺酮(100 mg/kg)和甲苯噻嗪(5 mg/kg)之混合物而將成年的雄性裸鼠麻醉。利用NARISHIGE裝置及具有26.5-規格針頭的WPI注射器,完成hMSCs的立體定向注射,注射座標分別為AP:-1 mm,ML:1 mm,DV:2 mm(相對於前囟點[AP]、中線[ML]和硬腦膜[DV])。注射速率為1 μl/min,注射時間為5分鐘,分別將以載體處理之hMSCs,及以封裝有Fe3O4的RDV處理之hMSCs細胞,注射到成鼠的右側腦和左側腦。在植入hMSCs後,使用臨床1.5-T.MR系統(Signa Excite,GE Healthcare,USA)進行MRI。
利用2%異氟醚進行氣體麻醉,將老鼠放置到內徑3.7 cm的自製共振線圈內,使用由供應商所提供的快速自旋回波脈衝(FSE-XL,Fast spin echo pulse)序列(TR/TE=4000/101.4 ms,矩陣大小=288×192)。切片厚度為0.8 mm帶有0.2 mm的缺口,且視野(FOV,field of view)為5×2.5 cm,全部的掃描時間為3分鐘20秒且激發次數(NEX)為8,由GE healthcare提供之工作站進行影像分析。
利用流式細胞儀(BD FACSCalibur),基於用於評估物質封裝效率之標準程序,而對封裝有FITC之RDV進行分析。亦利用流式細胞儀分析已加載,根據其標準步驟去發展,內被置入封裝有FITC的RDV之細胞,以評估RDV的藥物遞送能力。
將細胞與20 μl的Lysotracker(Invitrogen公司)(1%存在培養基中)進行培育,然後在4%仲甲醛/400 mM蔗糖/PBS中被固定,利用共軛焦顯微鏡(Leica)觀察及取得封裝有螢光團之RDV進入細胞內。
將hMSCs加載以RDV,並將其分別生長於正常、生脂和生骨樣培養基中。正常培養基係由低葡萄糖杜氏修改英格氏培養基(DMEM;Gibco)輔以10% FBS(HyClone,Logan,UT)、4 mM左旋麩醯胺酸(L-glutamine)、100 U/ml盤尼西林和100 μg/ml鏈黴素(Sigma-Aldrich)所組成。生脂性培養基係由高葡萄糖DMEM輔以異丁基-1-甲基黃嘌呤(0.5 mM,IBMX;Sigma-Aldrich)、地塞米松(1 mM,Sigma-Aldrich)、胰島素(10 ngmLS1,Sigma-Aldrich)、吲哚美辛(50 mM,Sigma-Aldrich)及FBS(10%)所組成。生骨性培養基係由α-MEM(GIBCO)輔以地塞米松(1 mM)、β-甘油磷酸酯(50 mM,Sigma-Aldrich)及抗壞血酸(50 mgmLS1,AsA;Sigma-Aldrich)所組成。
紅血球(RBCs)會隨著病狀(pathologies)的不同而有大小上的差異,但大部分的直徑是7.5-8微米。當RBCs和等體積的PBS混合(200 μl:200 μl)時,RBCs的形態近似完好無缺。當和等體積的dd H2O混合(200 μl:200 μl),RBCs看起來大上一圈且細胞膜表面變得比較粗糙。當和Ca2+-EDTA及雙去離子水以200 μl:50 μl:50 μl:100 μl(RBCs:1M CaCl2:390 mM EDTA:H2O;如圖一所示)比例混合時,RBCs開始形成芽體,當Ca2+-EDTA濃度上升到200 μl:70 μl:70 μl:60 μl(RBCs:1M CaCl2:390 mM EDTA:H2O;如圖一D),該芽體形狀變得最為豐富。該芽體會隨著RBC變成囊泡而消失。若Ca2+-EDTA濃度上升至高於200 μl:80 μl:80 μl:40 μl(RBCs:1M CaCl2:390 mM EDTA:H2O;如圖一所示)之比例,則RBCs會溶解。根據本發明,將從紅血球衍生得具有亞微米直徑之囊泡定義為RDV。
藉由粒度分析儀估計RDV大小為213.4至218.5 nm,平均直徑為215.9 nm。圖二為RDV的透式電子顯微鏡圖片。測量到存在全部受測試的RDV內之固有含量為大約1.213 nmoles每165 μg蛋白質。鐵之存在可增加TEM影像之對比,因此有助於在TEM下觀察RDV。利用TEM量測RDV的直
徑為大約259 nm,會比粒度分析儀估計的還要稍大些。如圖13所示,RDV的直徑分佈為213.4至218.5 nm,平均直徑為215.9 nm,這些奈米大小之RDV已經足夠小到能被細胞吸收。
為了證明RDV能夠將外來物質攜帶進入細胞內,遂將Fe3O4(氧化鐵為二價、三價的鐵)(磁石)封裝入RDV中,然後將其遞送進入人體骨髓間充質幹細胞(hMSC)內。如圖三所示hMSC沈澱物的MRI影像,上圖表示試管內囊泡的影像,下圖是位於試管底層之沈澱物的影像。當試管底部有黑點時表示偵測到標示有氧化鐵的細胞。試管1-4分別含有經載劑、RDV、Fe3O4及RDV-加-Fe3O4處理之hMSCs的細胞沉澱。試管5含有經封裝有Fe3O4的RDV處理之hMSCs的細胞沉澱。只有經封裝有Fe3O4的RDV處理過之細胞能夠被成像,顯現出一非常黑的點,表示Fe3O4已成功封裝入到RDV內,且經封裝有Fe3O4的RDV能被hMSCs吞沒。載劑係由100 μl低滲透壓溶液和2 ml DMEM組成。
流式細胞儀
將RDV封裝以各種不同的螢光分子。圖五左手邊圖形的A峰代表RDV內並沒有封裝FITC,而圖形右手邊的B峰表示已成功地封裝有FITC之RDV。當RDV內含有封裝的FITC
時,則會發現螢光強度向右位移,此螢光強度的增加確定RDV內封裝有FITC。
圖六係藉由流式細胞儀進行定量量測被細胞攝入之RDV。圖形左手邊的A峰表示與其中並未封裝有FITC之RDV進行培養的NIH3T3細胞,而圖形右手邊的B峰表示經封裝有FITC之RDV處理過的NIH3T3細胞。該內含封裝有FITC之RDV的細胞呈現其螢光強度高峰會向右移,這表示FITC藉由RDV被成功遞送進入NIH3T3內。
共軛焦顯微鏡
將FITC封裝於RDV內,來顯示RDV可當做用於細胞內造影及追蹤之物質的載體。其中,當RDV被B16或hMSCs細胞吞沒時,可以藉由共軛焦顯微鏡內觀察到FITC和FAM的綠色螢光,而Lysotracker被觀察到在溶酶體(lysosomes)上有紅色的螢光產生。在全部案例中,影像重疊同時顯示出RBC衍生囊泡的綠色螢光和溶酶體的紅色螢光。如圖7顯示在經封裝有FITC之RDV處理後的B16細胞(上圖)和hMSCs(下圖)的圖像。A:FITC螢光染色細胞:B:Lysotracker螢光染色細胞;C:A和B影像重疊。
圖8顯示在經封裝有FITC-紫杉醇之RDV處理後的B16細胞(上圖)和hMSCs細胞(下圖)的圖像。A:FITC螢光染色細胞;B:Lysotracker螢光染色細胞;C:A和B影像重疊。FITC-
紫杉醇攝入B16細胞(上圖)和hMSCs細胞(下圖)中,暗示抗癌症藥物可以封裝於RDV內當作一種藥物傳送系統。
由FAM-dsDNA、FAM-ssDNA和FAM-siRNA被攝入B16和hMSCs細胞內,顯示RDV可能應用於當作基因治療之遞送系統。圖9列示經封裝有FAM-dsDNA之RDV處理後的B16細胞(上圖)和hMSCs細胞(下圖)。A:FITC螢光染色細胞;B:Lysotracker螢光染色細胞;C:A和B影像重疊。siRNA為由有義與反義股組成的雙股RNA。做為陰性對照組,該siRNA不會靶定任何蛋白質。
圖10列示經封裝有FAM-ssDNA之RDV處理後的B16細胞。A:FITC螢光染色細胞;B:Lysotracker螢光染色細胞;C:A和B影像重疊。圖11列示經封裝有FAM-siRNA之RDV處理後的B16細胞(上圖)和hMSCs細胞(下圖)。A:FITC螢光染色細胞;B:Lysotracker螢光染色細胞;C:A和B影像重疊。
為了測試RDV做為活體內細胞追蹤試劑的實用性,將已經以封裝有氧化鐵之RDV處理過的hMSCs注射入成鼠腦之左側邊。並將經過載劑處理之細胞注射入成鼠腦的右側邊當作對照組。然後將小鼠用MRI進行造影。如圖4所示,只有左邊的腦部有顯現出黑點影像(以圈點表示)。
為了測試RDV之生物安全性,hMSCs在經過單獨RDV處理後進行檢測,以研究是否有危害到其分化潛能。如圖12所示為利用顯微鏡(Olympus)觀察經組織化學染色的細胞。脂肪細胞利用油紅進行染色以偵測細胞內呈紅色之脂質。骨細胞利用固藍(Sigma)進行染色以偵測細胞內呈紫色的鹼性磷酸酶。油紅色和固藍色的結果分別顯是在圖上和圖下。經過載劑處理過之hMSCs在正常培養基上成長,並不會顯現出任何紅油染色(上圖,A),表示在正常培養基中沒有細胞會分化成為脂肪細胞。經過載劑處理過之hMSCs在誘導脂肪細胞分化的培養基生長,會分化成脂肪細胞(上圖,B);同樣地,經過RDV處理之hMSCs在誘導脂肪細胞分化的培養基生長,也能夠分化成脂肪細胞(上圖,C)。
在固藍染色下,經過載劑處理的hMSCs在正常培養基上成長,並不會顯現出任合固藍染色(下圖,A),表示沒有細胞會分化成為骨細胞。經過載體處理之hMSCs在生骨培養基中生長可顯現出固藍染色,表示幹細胞會在生骨培養基中被誘導分化成骨細胞(下圖,B);同樣地,經RDV處理之hMSCs在生骨培養基中亦顯現出固藍染色,表示幹細胞在生骨培養基中會被誘導分化成骨細胞。上述結果表示RDV內在化並不會影響hMSCs分化成脂肪細胞(上圖)及骨細胞(下圖)的可能性,且RDV並不會影響細胞的生存能力(資料並未顯示)。
下述的描述及圖示已揭示本創作之較佳實施例,必須瞭解到各種增添、修改和取代可能使用於本創作較佳實施例,而不會脫離如所附申請專利範圍所界定的本創作原理之精神及範圍。熟悉該技藝者將可體會本創作可能使用於很多形式、結構和材料的修改。因此,本文於此所揭示的實施例於所有觀點,應被視為用以說明本創作,而非用以限制本創作。本創作之範圍應由後附申請專利範圍所界定,並涵蓋其合法均等物,並不限於先前的描述。
圖1A-1E顯示紅血球在各種溶液內的顯微影像。A:RBCs於磷酸鹽緩衝食鹽水溶液(PBS)中;B:RBCs於等體積的水中;C:RBCs於較低濃度的Ca2+-EDTA中,開始形成芽體(如箭頭所示);D:RBCs於適當的Ca2+-EDTA濃度內,形成豐富的RDV;E:大量的紅血球溶解在高濃度的Ca2+-EDTA內,尺規線:50 μm。
圖2顯示RDV在穿透式電子顯微鏡的影像。箭頭所指為具有直徑259 nm的RDV,尺規線:500 nm。
圖3為以人體骨髓間充質幹細胞(hMSCs)之MRI影像顯示RDV傳遞外源物質鐵進入幹細胞中。上圖顯示:試管垂直影像;下圖顯示:在試管底部球狀細胞的影像。1:經載體處理;2:經RDV處理;3:經Fe3O4處理;4:經RDV-加-Fe3O4處理;5:經封裝有Fe3O4之RDV處理。
圖4為小MRI影像,顯示位於腦左側上經磁性標定的hMSCs(以圓圈標示出)。
圖5為流式細胞儀圖譜,顯示其中內有(B)或無(A)封裝FITC之RDV的螢光強度。
圖6為流式細胞儀圖譜,顯示有與(B)或未與(A)封裝有FITC之RDV培養的NIH3T3細胞。
圖7為顯示細胞以封裝有FITC之RDV處理後的共軛焦顯微鏡影像。上圖顯示:B16細胞,下圖顯示:hMSCs。A:FITC螢光染色之細胞;B:Lysotracker螢光染色之細胞;C:A和B影像重疊,尺規線:25 μm。
圖8為顯示細胞以封裝有FITC-紫杉醇之RDV處理後的共軛焦顯微鏡影像。上圖顯示:B16細胞,下圖顯示:hMSCs。A:FITC螢光染色之細胞;B:Lysotracker螢光染色之細胞;C:A和B影像重疊,尺規線:25 μm。
圖9為顯示B16細胞以封裝有FAM-dsDNA之RDV處理後的共軛焦顯微鏡影像。上圖顯示:B16細胞,下圖顯示:hMSCs。A:FITC螢光染色之細胞;B:Lysotracker螢光染色之細胞;C:A和B影像重疊,尺規線:25 μm。
圖10為顯示B16細胞以封裝有FAM-ssDNA之RDV處理後的共軛焦顯微鏡影像。A:FITC螢光染色之細胞;B:Lysotracker螢光染色之細胞;C:A和B影像重疊,尺規線:50 μm。
圖11為顯示細胞以封裝有FAM-siDNA之RDV處理後的共軛焦顯微鏡影像。上圖顯示:B16細胞,下圖顯示:hMSCs。A:FITC螢光染色之細胞;B:Lysotracker螢光染色之細胞;C:A和B影像重疊,尺規線:50 μm。
圖12為顯示以組織化學染色之細胞的成像。上圖顯示:油紅染色法;A:經過載體處理之hMSCs生長於正常培養基;B:經過載體處理,生長於生脂肪性(adipogenic)培養基;C:經過RDV處理,生長於脂肪生成性培養基;下圖顯示固藍(fast blue)染色法;A:經過載體處理之hMSCs生長於正常培養基;B:經過載體處理,生長於生骨性(osteogenic)培養基;C:經過RDV處理,生長於生骨性培養基。尺規線:50 μm。
圖13A為顯示RDV直徑分佈之直方圖。
圖13B為顯示圖13A之RDV直徑測量值的圖表。
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Claims (18)
- 一種用於製備經分離的無細胞骨架紅血球微囊之方法,其中該紅血球微囊(Red blood cell-derived vesicles;RDV)具有直徑不大於500 nm,其包含下列步驟:a.從血液樣品製備出紅血球細胞;b.準備1 M CaCl2、390 mM EDTA及雙去離子水(ddH2O);以及c.將RBC、CaCl2、EDTA和ddH2O根據下列體積比例混合以得到含有RDV的混合物:i. CaCl2:EDTA=1:1;ii. RBC體積為CaCl2的2.5~5倍;iii. ddH2O體積為RBC和CaCl2:EDTA的總合之差值。
- 如申請專利範圍第1項所述之方法,其中步驟(c)係在低於50℃之溫度下完成。
- 如申請專利範圍第1項所述之方法,其進一步包含將混合物離心以收集得該經分離的RDV。
- 如申請專利範圍第1項所述之方法,其中該RDV具有之平均直徑係小於350 nm。
- 如申請專利範圍第1項所述之方法,其進一步包含將該紅血球微囊(RDV)和含有欲被封裝之外源性物質之溶液,混合於低滲透緩衝溶液(Na2HPO4/NaH2PO4,20 mM,pH 8)中,而製備一包含有經封裝之外源性物質之經分離的RDV。
- 一種於活體外將物質遞送進入細胞的方法,其包含下列步驟:a.將細胞和一其中包含有被封裝的外來物質之如申請專利範圍第1項所述之方法製備得之經分離的紅血球微囊(Red blood cell-derived vesicles;RDV)接觸;以及b.令該細胞將RDV內化而得到RDV-內化細胞,藉此將物質遞送進入細胞內,其中該紅血球微囊為一種無細胞骨架囊泡,且具有直徑不大於500 nm。
- 如申請專利範圍第6項所述之方法,其中該RDV具有直徑不大於350nm。
- 如申請專利範圍第6項所述之方法,其中該物質為至少一種選自螢光團、核酸、胜肽、多醣、超順磁性化合物及治療試劑者。
- 如申請專利範圍第6項所述之方法,其中該細胞為幹細胞且該物質為超順磁性化合物。
- 如申請專利範圍第9項所述之方法,其進一步包含利用磁共振成像(MRI)來追蹤該RDV-內化幹細胞內的超順磁性化合物。
- 如申請專利範圍第9項所述之方法,其中該幹細胞及RDV之來源為自體移植的。
- 如申請專利範圍第9項所述之方法,該超順磁性化合物為Fe3O4。
- 如申請專利範圍第6項所述之方法,其中該細胞為至 少一種選自初級細胞、癌細胞和幹細胞者。
- 如申請專利範圍第6項所述之方法,其中該細胞和RDV之來源為自體移植的。
- 一種如申請專利範圍第5項所述之方法製備得之經分離的RDV,其包含被封裝之外源性物質,其中該RDV為一種無細胞骨架囊泡,且具有直徑不大於500 nm。
- 如申請專利範圍第15項所述之經分離的RDV,其中該外源性物質為至少一種選自螢光團、核酸、胜肽、多醣、超順磁性及治療試劑者。
- 如申請專利範圍第15項所述之經分離的RDV,其中該包含有被封裝之外源性物質的RDV係被遞送進入非巨噬細胞內。
- 如申請專利範圍第15項所述之經分離的RDV,在其細胞膜表面上並無任何修飾。
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