表面修飾麥胚凝集素之心磷脂微脂粒包覆神經生長因子與薑黃素在穿透血腦屏障和抑制β-amyloid誘發SK-N-MC細胞凋亡的效應

NGF and Curcumin Encapsulated into Wheat Germ Agglutinin Grafted Cardiolipin Liposomes for Transporting across the Blood-brain Barrier and Counteracting the Degeneration of SK-N-MC Cells Induced Apoptosis by β-amyloid Peptide

林敬鈞(Chun Ching Lin)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2013年)

摘要

為改善薑黃素(curcumin)難溶於水與在生物體內利用度低，與神經成長因子(nerve growth factor, NGF)在腦部的低穿透率情形，利用與β-amyloid (Aβ)具有高親和性的磷脂質cardiolipin (CL)混和1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)與膽固醇製備微脂粒作為藥物載體，包覆curcumin與NGF，並在表面改質麥胚凝集素(wheat germ agglutinin, WGA)，欲藉此增加腦微血管內皮細胞對粒子的攝入量，使包覆curcumin與NGF粒子能夠順利穿透腦部，並標靶至阿茲海默氏症腦部神經退化區域。使用人類腦微血管內皮細胞、周細胞及星形細胞共培養建立體外血腦屏障(blood−brain barriers, BBB)模型探討NGF與curcumin穿透率，並利用fibrillar β-amyloid peptide-(1-42) (Aβ1-42)誘發人類神經胚胎腫瘤細胞株SK-N-MC凋亡，模擬神經退化情形，建立一個阿茲海默氏症體外模型。實驗結果顯示，隨著CL比例的增加，微脂粒的粒徑增加、電位下降。粒子經WGA改質後的平均粒徑亦隨之上升，比改質前粒徑增加約6 nm，以各組微脂粒進行體外血腦屏障模型穿透實驗中，加入麥胚凝集素改質後的粒子，發現對NGF來說，改質後的微脂粒穿透率為未改質粒子的1.71倍至2.52倍，對curcumin來說，改質後的微脂粒穿透率為未改質粒子的1.82倍至2.64倍，顯示經WGA改質後會提升微脂粒的穿透率。包覆NGF與curcumin的微脂粒抑制fibrillar Aβ1-42引發SK-N-MC凋亡實驗結果顯示，細胞存活率方面，加入包覆curcumin的心磷脂微脂粒(cardiolipin liposomes, CLL)高於free curcumin，且隨著CL含量的增加而上升，同時包覆curcumin與NGF的CLL具有最高存活率。以包覆螢光物質(FITC)的微脂粒分別與腦微血管內皮細胞及SK-N-MC共培養，免疫螢光染色圖顯示，WGA改質能提高腦微血管內皮細胞對粒子的攝入，並且cardiolipin能賦予微脂粒具有標靶Aβ1-42的特性。上述結果顯示curcumin與NGF經由WGA表面修飾的心磷脂微脂粒包覆後，可有效提升BBB的穿透率，抑制β-amyloid誘發SK-N-MC細胞凋亡，因此，以WGA改質心磷脂微脂粒藥物載體未來可有效應用於阿茲海默氏症的臨床治療。

關鍵字

心磷脂 ； 麥胚凝集素 ； 阿茲海默氏症 ； 薑黃素 ； 微脂粒

並列摘要

In order to improve the hydrophilic and poor bioavailability of curcumin and the low permeability of nerve growth factor (NGF) in the brain, liposomes were used to be drug carriers to encapsulate curcumin and NGF. The liposomes content with cardiolipin were provided with high affinity for β-amyloid (Aβ). The surface of liposomes was modified with wheat germ agglutinin (WGA) to increase cellular uptake. Investigating the curcumin NGF permeability of the in vitro blood brain barriers (BBB) model was established by human brain microvascular endothelial cells (HBMECs), human pericytes (HPs) and human astrocytes (HAs) on the transwell, and the protective effect of curcumin and NGF against the in vitro Alzheimer’s disease (AD) neurodegenerative model was established by SK-N-MC neuroblastoma cell line induced apoptosis by Aβ1-42 fibrils. The results indicated that an increase in molar percentage of cardiolipin enhanced the average diameter and zeta potential. The average diameter increased approximately 6 nm compared to unmodified liposomes. HBMECs were incubated with fluorescent liposomes, and the fluorescent images indicated that WGA could enhance cellular uptake. SK-N-MC cells were incubated with fluorescent liposomes and fibrillar Aβ1-42, and the fluorescent images indicated that the liposomes content with cardiolipin could bind on the fibrillar Aβ1-42. These evidences suggested that cuecumin and NGF encapsulated into WGA grafted cardiolipin liposomes can enhance the permeability against BBB and attenuate β-amyloid induced apoptosis in SK-N-MC cells. Therefore, WGA/curcumin-NGF-cardiolipin liposomes can be a potential drug in Alzheimer’s disease therapy for clinical application.

並列關鍵字

cardiolipin ； wheat germ agglutinin ； Alzheimer’s disease ； curcumin ； liposomes

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