探討單股核酸包覆奈米粒子於吞噬機制及細胞標定之應用

Abstract

一般認為，帶正價電之粒子相較於不帶電或是帶陰電性之粒子容易吸附於細胞膜上被細胞所吞噬，在過去基因傳輸系統的研究中，確實已證實帶正電的粒子或載體效力較佳。然而，透過YC Chung等學者研發出帶陰電性單股核酸之基因載體，能有效達到轉殖之效能，因此，本研究將建立陰電性單股核酸載體於細胞吞噬之機制，藉由添加不同路徑之抑制劑，得知，載體能經由兩種以上之路徑進入細胞，然而，只有caveolar及macropinocytosis pathway能達到基因轉殖之效能，一旦確認載體之路徑不僅能改善未來基因載體之製備過程，亦能避免載體進入lysosomes而大幅提升載體之轉殖效能。 此外，本研究藉由單股核酸包覆於基因轉殖載體之方法，結合工研院提供表面帶有不同官能基之氧化鐵粒子作為模板，製備出以單股核酸組裝外層之陰電性氧化鐵粒子。由於氧化鐵本身特性影響，在包覆多層聚電解質時，顯示細胞毒性會受到所使用陽離子層所調控，經單股核酸包覆後能同時降低細胞毒性並被細胞吞噬；此包覆技術運用至氧化鐵上，可直接製備出不須純化，進一步修飾氧化鐵粒子。此外，將單股核酸包覆氧化鐵粒子與吞噬機制結合，得知經單股核酸包覆後之粒子，即可藉由不同之吞噬路徑進入到細胞內。 因此，本研究成功以單股核酸包覆氧化鐵粒子作為模板，未來可利用核酸互補的性質作為連結標的分子和氧化鐵的架橋，發展出磁光合一的功能或是帶有標的功能之氧化鐵粒子，預期將可應用於臨床磁振造影且同時具有螢光標定追蹤癌細胞之功能。

Generally, particle uptake by cells is mediated by electrostatic interactions of positively charged particles with negatively charged cellular surface, so the cationic polyplexes showed efficiently cellular transfection. However, YC Chung et al. developed a new complex with high transfection efficiency by assembling negatively charged single-stranded oligonucleotides with polycation/DNA complexes. Therefore, this project investigated the cellular uptake pathway of single-stranded oligonucleotides with polycation/DNA complexes. The results of different inhibitors showed these complexes can cellular uptake by several pathways. However, only the caveolae and macropinocytosis pathway would influence gene expression. Furthermore, iron oxide particles will be investigated by single-stranded oligonucleotide-assembled technique. The results indicate the oligonucleotide-coated iron oxide particles could be uptaken and present low cytotoxicity. This technique of oligonucleotide-assembled iron oxide particles can easily manufacture and without further purification. To integrate the oligonucleotide-coated iron oxide particles and cellular uptake pathway, the results also shows the oligonucleotide-coated iron oxide particles could be internalized by several pathways. Finally, this project successfully develop a multifunctional iron oxide platform by conjugating oligonucleotides, and also develop the ligand or fluorescent probe by nucleic acid base-paired interaction. Therefore, the multifunctional iron oxide particles can be applied to MRI or fluorescent target to track tumor in vivo.