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  • 學位論文

葉酸修飾之脂質奈米膠囊之製備與其對乳癌細胞的標的性

On the Preparation of Folate-modified Lipid Nanocapsules and Their Targeting Ability towards Breast Cancer Cells

指導教授 : 何國川

摘要


本研究主要著重於脂質奈米膠囊的製備及其表面修飾葉酸,作為具有生物相容性的載藥粒子,並且探討葉酸修飾之脂質奈米膠囊對於乳癌細胞上大量表現的葉酸受體之作用,以達到增進脂質奈米膠囊對乳癌細胞的標定效果。 脂質奈米膠囊是利用相轉換溫度法合成,隨著溫度的提升乳膠系統從o/w經過一個相轉換區 (Phase transitional zone) 後變成w/o/w,在進入相轉換區後加入大量冷卻水以得到脂質奈米膠囊產物。本研究目的在於將葉酸分子修飾上脂質奈米膠囊表面,所以先利用胺丙基三乙氧基矽烷與脂質奈米膠囊進行縮合反應讓奈米膠囊表面帶有胺基,接下來胺丙基三乙氧基矽烷修飾之脂質奈米膠囊與葉酸分子之間,則是利用葉酸活性酯 (NHS/DCC方法) 與胺基所形成的胜肽鍵加以鍵結。 接著討論經葉酸分子修飾後的脂質奈米膠囊其各項性質,從傅立葉轉換紅外線 (FTIR) 光譜矽-氧-碳及碳氧雙鍵特徵峰的出現,可以確認胺丙基三乙氧基矽烷與脂質奈米膠囊的縮合反應成功以及葉酸分子與胺丙基三乙氧基矽烷修飾之脂質奈米膠囊之間的鍵結。在粒徑分佈、穿透式電子顯微鏡 (TEM)中可看出修飾過葉酸後脂質奈米膠囊的粒徑從10 nm增加至40 nm左右,且沒有聚集現象產生。 為了測試葉酸修飾之脂質奈米膠囊其細胞毒性以及標定效果,本研究將不同濃度葉酸修飾之脂質奈米膠囊與正常的纖維母細胞 (3T3) 一同培養,用來測試所合成奈米膠囊的細胞毒性,結果顯示纖維母細胞對於葉酸修飾之脂質奈米膠囊可容忍的最大濃度為55 μL/mL。最後藉由流式細胞儀及雷射掃瞄共軛焦顯微鏡來測試其標定效果。在流式細胞儀及共軛焦顯微鏡的數據中均可以看出修飾上葉酸的脂質奈米膠囊能提升其對乳癌細胞(BT-20) 的標定效果。

並列摘要


In this study, lipid nanocapsules (LNCs) modified with folic acid (FA), denoted as folate-LNCs, were synthesized and their interactions with the folate receptors over-expressed on the surface of the breast cancer cells are discussed. The folate-LNCs were synthesized with the expectation of improving the targeting ability of LNCs towards the breast cancer cells. The LNCs were synthesized by the phase transition temperature method. During heating process, emulsion system changed from o/w and passed through the phase transitional zone to w/o/w. LNCs were produced by adding large amount of cooling water at the phase transitional zone. To synthesize folate-LNCs, the surface of the LNCs was first modified with a layer of 3-aminopropyltriethoxysilane (APTES) to generate amino groups on the surface of LNCs by condensation reaction. Thereafter, folic acid was conjugated to APTES-LNCs through the formation of the peptide bond by the N-hydroxysuccinimide ester of folic acid (NHS/DCC method) and amino group. The Si-O-C and C=O peaks in the FTIR spectrum were observed, indicating the folate-LNCs were successfully synthesized. Besides, the particle size increased from 10 to 40 nm after FA was conjugated to LNCs, evidenced by zeta-sizer and TEM. No aggregation was observed for the LNCs and folate-LNCs. In order to examine the biocompatibility of the folate-LNCs, the 3T3, fibroblast cells were cultured with various concentrations of folate-LNCs. It was found that the maximum tolerance concentration of the folate-LNCs to fibroblast cells was 55 μL/mL. On the other hand, the targeting ability of the folate-LNCs to the human breast cancer cells, BT-20, was examined by the flow cytometry and confocal microscopy techniques. The results showed that the targeting ability of the LNCs to breast cancer cells was improved by conjugating folic acid.

參考文獻


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