利用同步輻射X光及中子散射技術研究混合長鏈及短鏈磷脂分子的平板微胞與去氧核醣核酸的交互作用及結構變化

Neutron and Synchrotron X-Ray scattering studies on the structure and Phase Behavior of Mixed Short-Chain/Long-Chain Bicelles and Their Interaction with DNA

楊博偉(Po-Wei Yang)

指導教授 ： 林滄浪

國立清華大學/原子科學院/工程與系統科學系/博士(2012年)

https://doi.org/10.6843/NTHU.2012.00322

摘要

不同於球狀囊泡型脂質與DNA自組裝形成的複合載體，在這份研究論文中，我們利用小角度散射技術並搭配穿透式電子顯微鏡影像研究探討圓盤狀微胞(Bicelle)與DNA所形成的三維複雜結構。所謂的圓盤狀微胞是由兩種鏈長不同的脂質(長鏈的DPPC與短鏈的diC7PC)在特定混合比例、溫度及濃度條件下自組裝形成的結構；而因為分子形狀不同的緣故，使得DPPC與diC7PC分別組成圓盤狀微胞的中央平面處與側邊曲率較大處。圓盤狀微胞在不同混合比例的結構以及特定比例(DPPC/diC7PC=3/1)隨溫度變化之相行為的基礎研究將先被探討。我們發現在這兩相混合系統中，短鏈的脂質數量增加時，其微胞結構將逐漸從多層球狀微胞漸進變化至小橢圓微胞。當長短脂質混合比為3且溫度由25°C逐漸增加至45°C，側邊的短鏈脂質因高溫而熔進平板區域，這使得單一分散的圓盤狀微胞也轉換至多層狀結構。為了與DNA形成複合體，圓盤狀微胞上的長鏈脂質分別由帶正電的DC-chol以及負電荷DPPG取代成為帶有電荷的平板微胞。首先，正電圓盤狀微胞透過電吸引力與負電DNA 形成的層狀聚集結構大小可藉由改變每一個正電圓盤狀微胞的電荷密度及DNA的長度與濃度來調控。這意味著此種新型的圓盤狀微胞與DNA的複合載體在製備上有尺寸可調整的優勢。由TEM影像可發現數個圓盤狀微胞也將匯聚成一個較大的平面以便於穩定地夾帶DNA於其中。而這樣的複雜體也被證實具有對抗高溫的穩定性。為了降低細胞毒性之緣故，負電荷的圓盤狀微胞也曾被用於與DNA形成複合載體。在足夠量多價陽離子的幫助下，這個類似於正電荷圓盤狀微胞與DNA的多層結構隨著變化不同二價離子的濃度經歷了有趣的相變行為。二價陽離子的凝縮效應顯著的被發現在DNA與負電荷的平板微胞之間。在二價陽離子增加到一臨界濃度時，所有DNA都被侷限在負電圓盤狀微胞的夾層中。若將離子持續增加，多餘的脂質將被釋出與周遭的二價離子形成多層的球狀結構。而其間距被發現隨著離子濃度增加而遞減。我們系統化地合成並搭配結構探討的研究，期盼此新型的圓盤狀微胞與DNA所形成的複合載體能提供新思維並應用於基因治療或藥物輸送的載體設計。

關鍵字

小角度散射 ； 圓盤狀微胞 ； 去氧核醣核酸 ； 基因治療

並列摘要

In this present work, the disk like micelles, called bicelle, composed by long-chain lipids (DPPC) and short-chain lipids (diC7PC) at the planar and the rim region, respectively, were used to form the complex with DNA in contrast to conventional vesicle based lipids-DNA complex. The fundamental research on the binary mixture of DPPC/diC7PC was first constructed, including the structure transformation in different mixture ratio and the phase transition upon the increasing temperature in the selected mixing ratio of DPPC/diC7PC=3/1. We then pack the DNA with the charged bicelles by doping cationic DC-chol or anionic DPPG. In the case of using cationic bicelles, self-assembly CB-DNA complexes were found to have the lamellar structure of alternating DNA-membrane. By turning the membrane charge density, the CB-DNA complex exhibited the benefits on its small size and tunable size with a constant DNA packing density. Moreover, it also showed the structure stability against the increased temperature. Subsequently, the DPPG doped bicelles were found to form the complexes with the DNA, called AB-DNA complex, only in the presence of sufficient calcium ions. Such the AB-DNA complex had the alternating membrane-ion-DNA lamellar structure which is similar with the CB-DNA complex. When calcium ions exceed a critical concentration, the DPPG MLV was found to coexistence with the AB-DNA complex since all the DNA was condensed in the AB-DNA complexes. The systematically demonstrated CB-DNA and AB-DNA complexes would provide a new stratagem to design new complexes which have distinct structure from previous vesicle-based lipids-DNA complexes.

並列關鍵字

SAS ； Bicelle ； DNA ； Gene therapy

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