利用單分子光譜技術探討脂雙層膜的膜相和膜曲度對脂質動力學的影響

Abstract

這個實驗利用螢光相關光譜 (FCS) 結合單分子光譜 (SMS) 的技術，研究奈米等級微脂體的脂質動力學。我們製備四種不同的微脂體：DPPC-SUV、DPPC-LUV、 DOPC-SUV、DOPC-LUV，並嵌入染料分子 DiD 於脂雙層作為反映膜狀態的觀察標的，這些可以幫助我們了解室溫下微脂體在不同的膜相 (DPPC 和 DOPC) 及膜曲度 (SUV 和 LUV) 下，是否會影響 DiD 的順反異構化生命期 (cis-trans isomerization lifetime) 、三重態生命期 (triplet state lifetime) ，和擴散係數 (translational diffusion coefficient)。實驗結果顯示這三種參數對於這兩種膜相皆有差異，另外發現 DiD 的三重態生命期在 DOPC 比在 DPPC 膜內長，可能是鬆散的排列使 DOPC 容易和極性分子形成氧氣屏障；DPPC 雖然被認為膜的質地較堅硬，氧氣也不易透入，但此結果的呈現可能可以歸咎於 DPPC 容易在表面產生缺陷 (defect)，反而使氧氣較易滲透。擴散係數的結果顯示無論在哪種膜相， DiD 在曲度大的微脂體中皆擴散較慢，推測可能是因為曲度大的微脂體較易使脂質分子排列不規則，使DiD 在其中的擴散變慢。

We developed a technique which combined fluorescence correlation spectroscopy (FCS) and single molecule spectroscopy (SMS) to investigate lipid dynamics in bilyer membranes. Due to the difficulty of direct observation of lipid molecules, we added cyanine dyes, 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate (DiD or DiIC18(5)) into four sets of samples including 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-small unilamellar vesicle (SUV), DPPC-large unilamellar vesicle (LUV), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)-SUV, and DOPC-LUV. DiD is widely used as a reflection of lipid packing. In this work, we focused on the effects of phase and curvature of membranes on three physical underlying parameters of DiD: cis-trans isomerization lifetime, triplet state lifetime, and translational diffusion coefficient. Under FCS-SMS measurement, we found that the results of these three parameters all presented differences between gel phase (DPPC) and liquid-crystalline phase (DOPC), the results of triplet state lifetime further showed that oxygen were possibly hindered by a thicker polar region formed by aqueous solution and head groups of lipids in DOPC. For DPPC, there may be some defects that provided easier permeation for oxygen. Translational diffusion coefficient presented interesting results that they became smaller along with the increasing of vesicle curvature. It can be inferred that smaller vesicles had more disordered arrangement of lipids than larger vesicles, which impeded the translational diffusion of DiD.