表面修飾Ln5-P4蛋白之固態脂質奈米粒子包覆視黃酸與神經成長因子驅動誘導式多能幹細胞分化成神經細胞之 界達電位與電泳遷移率

Effect of nerve growth factor and retinoic acid in Ln5-P4 peptide-grafted solid lipid nanoparticles on electrophoretic mobility and zeta potential of induced pluripotent stem cells during differentiating toward neurons

施黃竣元(Chun-Yuan Shih Huang)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

本研究以雙層乳化法(double emulsion technique)製備固態脂質奈米粒子(solid lipid nanoparticles, SLNs)，於脂質內包覆疏水性藥物視黃酸(retinoic acid, RA)與親水性藥物神經成長因子(nerve growth factor, NGF)，SLNs表面以1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)與N-hydroxysuccinimide (NHS)化學交聯方式將PPFLMLLKGSTR motif (Ln5-P4)鍵結於SLNs表面上，用以標靶誘導式多能幹細胞(induced pluripotent stem cells, iPSCs)膜上之α3β1 integrin，使SLNs更有效地被iPSCs攝入，並以高效能毛細管電泳儀分析SLNs與分化中細胞的電泳遷移率及界達電位效應。結果顯示出，隨著蜜蠟(beeswax, BW)含量上升時，平均粒徑下降，RA與NGF藥物包覆率增加，分別達82.7±4.32%、21.2±0.95%，降低RA與NGF釋放速率。製備SLNs過程中，隨著卵磷脂比例增加，造成SLNs粒徑上升、電泳遷移率絕對值與界達電位絕對值上升。此外，透過免疫螢光染色圖證實Ln5-P4/RA-NGF-SLNs能透過α3β1 integrin的辨識進入細胞，顯著地提升iPSCs對藥物攝入量，亦證實七天後的培養，iPSCs可以朝神經分化並表現出β-tubulin III。隨著誘導分化時間拉長，SLNs刺激分化中的iPSCs帶更多的負電，使電泳遷移率絕對值、界達電位絕對值及固定電荷密度絕對值上升。最後，以流式細胞儀對anti-SSEA-1和anti-β-tubulin III雙螢光染色分析，實驗結果顯示Ln5-P4/RA-NGF-SLNs與iPSCs進行共培養的結果顯示細胞達93.72%神經分化比例。

關鍵字

誘導式多能幹細胞 ； 界達電位 ； 電泳遷移率 ； 固態脂質奈米粒子 ； 視黃酸 ； Ln5-P4

並列摘要

Nerve growth factor (NGF)- and retinoic acid (RA)-loaded solid lipid nanoparticles (SLNs) were modified with PPFLMLLKGSTR motif (Ln5-P4) (Ln5-P4/RA-NGF-SLNs) for differentiation of induced pluripotent stem cells (iPSCs) toward neurons. Internalizations of Ln5-P4/NGF-RA-SLNs by iPSCs were examined by immunochemical staining to demonstrate the interactions between the nanoparticles and expression of α3β1 integrin. The results revealed that an increase in beeswax (BW) weight percentage reduced the average diameter, enhanced entrapment efficiency of RA and NGF, and extended elease rate of RA and NGF. An increase in lecithin weight percentage enhanced the particle size, absolute value of electrophoretic mobility, and zeta potential. In addition, the results of immunofluorescence demonstrated Ln5-P4 on RA-NGF-SLNs promoted the uptake amount of NGF and RA by iPSCs and proved iPSCs cultured with SLNs can differentiate toward neurons and express β-tubulin III after 7 days. Moreover, a longer inductive time with Ln5-P4/RA-NGF-SLNs produced a higher negative charge of differentiating iPSCs toward neurons. Ln5-P4/RA-NGF-SLNs could stimulate endocytosis of iPSCs via recognition of α3β1 integrin and decreased the electrophoretic mobility and zeta potential of iPSCs during neuronal differentiation. Finally, the flow cytometry analysis indicated that Ln5-P4/RA-NGF-SLN cultured with iPSCs can express 93.72% β-tubulin III. Based on our findings, we conclude that Ln5-P4/RA-NGF-SLN can be a promising delivery system.

並列關鍵字

induced pluripotent stem cells ； zeta potential ； electrophoretic mobility ； solid lipid nanoparticles ； retinoic acid ； Ln5-P4

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