利用可食性化合物修飾已包埋薑黃素之微脂體以增進其穿透血腦障蔽能力

Abstract

薑黃素可瓦解類澱粉β-胜肽堆積，在體內體外實驗中都證實其對腦部具有保護功效。然而，因血腦障壁 (Blood brain barrier, BBB) 緊密的結構和薑黃素生物可利用率低，使腦部吸收薑黃素非常困難，導致在臨床上應用有限。微脂體為奈米載藥系統，由雙層磷脂質組成，可包埋薑黃素，透過表面修飾可接上標的性物質。過去常使用聚乙二醇 (poly(ethylene glycol), PEG) 和運鐵蛋白 (Transferrin) 製備載體，提升載體在血液運輸的穩定度和標的運輸至血腦障壁，然而，PEG會堆積在生物體內無法分解，且會引發過敏反應，因此，找到取代材料非常重要。唾液酸 (Sialic acid, SA) 為可生物分解的材料，具有高度親水性特質，可於載體外形成水層，提高穩定性，做為替代PEG的材料，然而目前還未應用於微脂體載藥系統。小麥凝集素 (Wheat germ agglutinin, WGA) 可當作標的血腦障壁的分子，其會和腦內皮細胞上的唾液酸結合，將載體運輸至腦內。本研究以最佳比例之膽固醇與卵磷脂，建構出包埋率高的奈米微脂體，接著，以可食性之唾液酸、聚唾液酸 (Polymerized SA, PSA) 和其氧化態各別作為架橋，連接小麥凝集素進行微脂體之表面修飾，並透過血腦障壁細胞平台證實載體穿透血腦障壁的運輸效率。 研究結果顯示，微脂體對於薑黃素的包埋率高達90.77%，其粒徑約為115奈米。熱重分析 (Thermogravimetric analysis, TGA) 結果確認SA、PSA和其氧化態皆成功鑲嵌於微脂體的磷脂雙層中，100℃下TGA曲線顯示WGA可當作唾液酸的保護層。整體來說，SA或PSA較其氧化型態可連接較多含量之WGA於微脂體表面上，其中，聚唾液酸並無顯著增加連接WGA於微脂體表面。經過模擬人體消化酵素處理後，超過40% WGA仍存在於載體表面，另外，傅立葉轉換紅外光譜 (Fourier transform infrared spectrum, FTIR) 在1241 cm-1和533-581 cm-1具有醯胺基訊號，表示唾液酸的羧酸基和WGA的胺基以共價鍵互相連接形成醯胺基，即使經過消化酵素處理，醯胺基訊號仍然存在，顯示在微脂體表面的唾液酸和WGA之鍵結可抵抗消化酵素的分解。掃描式電子顯微鏡顯示經過消化酵素處理後，表面修飾之載體仍具有球體型態且有清楚的輪緣。另外，經過表面修飾和消化酵素處理之載體較未經過表面修飾且未經過消化酵素處理之載體具有較高穿透BBB之效率。本研究結果證實載體表面修飾具有提升微脂體運輸植化素穿透BBB之效率以達到保健腦部之潛力。

Curcumin has been proven to be able to effectively improve brain diseases, such as Alzheimer’s disease. However, low bioavailability of curcumin and blood brain barrier (BBB) limit its clinical uses. The development of new delivery system to improve transport of curcumin crossing the BBB appears to be a promising strategy for prevention or even treatment of brain diseases. Past researches focused on a strategy of using poly (ethylene glycol) (PEG) and transferrin to prolong the survival of drug in blood and facilitate brain drug delivery system. However, limitations of PEG including synthesis procedure, immunological response, and accumulation in tissues have been observed. In this study, we used alternative sialic acid (SA), polysialic acid (PSA), oxidized form of SA/PSA and wheat germ agglutinin (WGA) to synthetize multifunctional nano-liposomes to facilitate the transport of curcumin across BBB. Results showed that curcumin was entrapped in liposome with high encapsulation efficiency (90.77%) and the vector had a size of about 115 nm in diameter. The thermogravimetric analysis (TGA) confirmed the successful presence of SA and PSA in the lipid bilayers of liposomes. TGA curves below 100 °C suggested that WGA acts as a protective layer for SA. In general, the liposomes modified by the un-oxidized SA or PSA could conjugate more WGA than that oxidized counterparts, and polymerization of SA did not increase WGA conjugation on the liposomes. More than 40% of WGA on the surface of modified liposomes remained after digestive enzymes challenge. The amide peaks at 1241 cm-1 and 533-581 cm-1 in the Fourier transform infrared (FTIR) spectrum of the modified liposomes indicated the covalent bonding between carboxyl group of SA and amine group of WGA. These characteristic peaks still existed after digestive enzymes challenge, demonstrating that the conjugation of WGA and SA on the modified liposome can resist the digestive enzymes to a certain extent. SEM images showed that the modified liposomes after digestive enzymes treatment were still spherical with clear rim, and the enzyme-treated vectors had a higher permeation rate crossing blood-brain barrier (BBB) endothelial cell monolayer than the un-modified liposome even without enzyme treatment. This study demonstrates how the surface modification can enhance the function of liposome as a phytochemical carrier for brain disease.