疊層組裝之奈米粒子於短鏈核苷酸遞送之研究

Abstract

有效率的遞送系統不論是在治療或是研究中皆是一個重要的課題，利用正價物質可藉由電性間的相互吸引將核酸物質包覆形成幾百奈米之複合體進行基因遞送，但藉由正價物質進行活體基因遞送時會遇到許多障礙因而遞送效率較差，且不能穩定存在於活體中。為了提高粒子在活體內的穩定度及維持粒子大小，在先前的研究中利用強親水性的聚合物聚乙二醇(PEG)修飾粒子表面使其能有效提高在血管中的滯留時間及穩定性。 2-胍基-乙基-氨基甲酸(GEC-Chol)為一以膽固醇為基礎之正價脂質，在先前的研究中發現其在與膽固醇混合形成約100 nm之奈米粒子，在細胞試驗中遞送線型DNA具有良好的遞送效率。同樣的在本實驗中利用異硫氰酸螢光素(FITC)標記之短鏈核苷酸用以追蹤短鏈核苷酸，發現在氮磷比為3時遞送短鏈核苷酸可以展現近乎100 %的遞送效率，但是在加入10 % PEG-DOPE時，遞送效率卻幾乎無法觀察到遞送的效率。為解決遞送效率降低的問題，本實驗在核酸物質與正價脂質形成複合體後再外插入PEG-DOPE，期望可以降低粒子大小同時提高穩定性，使其更適合活體遞送。實驗結果證明使用後插入PEG-DOPE方法之粒子具有較小的粒徑、較高的穩定度。同時在流式細胞儀及螢光顯微鏡的分析中皆可以測定到帶有FITC之短鏈核苷酸即使在10 % PEG-DOPE依然不會影響核酸物質的遞送效率。為了在活體中容易觀察，因此在粒子中加入量子點，再經由共軛焦顯微鏡及洋菜膠電泳跑膠分析後，證明加入量子點不影響短鏈核苷酸的包覆。在活體實驗中發現，這樣的粒子在各組織中都有看到分佈的量子點，且這樣的粒子在體內中不會引發發炎反應，具有相當高的生物相容性。 本論文提出之以後混方式形成之粒子在有聚乙二醇存在時具有高度遞送效率，且在活體試驗中未明顯產生免疫反應，且在含有20 % PEG-DOPE之粒子可降低非專一性的遞送，綜合以上結果顯示，以後混方式形成之粒子比短鏈核酸與正價脂質直接混合之粒子更適合作為體內核酸藥物之載體，未來可同時加入藥物或是專一性遞送的導向性分子，使其成為具有專一性活體治療及活體影像之多功能奈米粒子。

The efficient gene delivery system is important both in therapeutic and laboratory research. The cationic materials can interact with negatively charged nucleic acids and assemble into several hundred nanometers in diameter complexes though the ionic interaction. However, there are several complications, for example, low delivery efficiency and poor stability, when applying these materials to in vivo gene delivery. In order to maintain the size of complexes and enhance the stability in vivo, previous studies have developed a strategy by using the highly hydrophilic polymer, polyethylene glycol (PEG), to modify the surface of particles hence improve the circulation time and stability. GEC-Chol (2-guanidino-ethyl-carbamic acid-cholesterol) is a cholesterol-based cationic lipid. Previous studies reveal that, when GEC-Chol is mixed with cholesterol, they assemble into 100 nm particles. These particles are highly efficient in linear DNA delivery in in vitro experiments. In this study, we used FITC-labeled oligonucleotides as tracers and found that when N/P ratio of ODN and lipid was 3, the high delivery efficiency was observed. But if we added 10 % PEG-DOPE into the particle formulation, the delivery efficiency decreased dramatically. To solve the conflict between stability and efficiency, we bring up a strategy, post insertion, as a resolution. Briefly, we inserted PEG-modified lipids, PEG-DOPE, to the complexes after the particle of ODNs and cationic lipids formed, expected to reduce the particle size and increase the stability for in vivo applications. The result revealed that post-insertion particle had smaller particle size and higher stability. Using fluorescence microscopy and flow cytometry, we could observe that even lipid particles containing 10 % PEG-DOPE still retained the high delivery efficiency of FITC-labeled oligonucleotides in vitro. In order to detected particles in vivo, we add high quantum yield nanoparticles, quantum dots (QDs), to indicate the distribution of post-insertion particles. After analysis by confocol microscopy and agarose gel, it demonstrated that addition of QDs did not affect oligonucleotide encapsulation. This kind of particles did not induce inflammation in vivo and had high biocompatibility. In this study, post-insertion particles containing 10 % PEG-DOPE have high delivery efficiency, and they do not induce obvious immune responses in vivo. Furthermore, if we elevate the PEG-DOPE ratio to 50 %, the nonspecific binding decreases evidently. The long and the short of it is that post-insertion particles are more suitable to be the carrier of nucleic acid drug in vivo than previous. In the future, we can add drugs or specific targeting molecules as tools for specific therapy and imaging in vivo.