以二元體形式之Indolicidin 應用於去氧寡核苷酸之輸送;The Use of Dimeric Indolicidin for Oligodeoxynucleotide Delivery Application

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Title:	以二元體形式之Indolicidin 應用於去氧寡核苷酸之輸送;The Use of Dimeric Indolicidin for Oligodeoxynucleotide Delivery Application
Authors:	黃詩淳;Huang,Shih-Chun
Contributors:	化學工程與材料工程學系
Keywords:	胜肽;去氧寡核苷酸;peptide;oligodeoxynucleotide
Date:	2016-08-30
Issue Date:	2016-10-13 12:47:51 (UTC+8)
Publisher:	國立中央大學
Abstract:	穿膜胜肽Indolicidin (IL)的毒性限制了其臨床應用性，為了增進生物適合性，本研究合成二元體形式之IL並討論其應用於輸送去氧寡核苷酸(ODN)的效果。藉由分別對Indolicidin其C端與N端接上一個半胱胺酸，胜肽得以形成雙硫鍵成為二元體，我們分別將其命名為ILC與CIL。以HPLC與MASS分析證明二元體之形成。這些二元體可以藉靜電複合的方式將之與ODN自組裝成奈米粒子。由MTT assay可以發現，相比於IL，CIL與ILC可以有效降低細胞毒性。利用動態雷射散射粒徑分析儀測量複合物之粒徑大小介於500至2000nm，在巨噬細胞可胞吞範圍內。在表面電位的表現上CIL與IL的電位可以隨著N/P比而提高其電位，但是ILC顯然地不易吸附於ODN，要到N/P 30以上才有正電位。以膠體電泳方式測試其包覆率，IL與CIL皆有良好的複合能力，可包覆ODN形成穩定的粒子，但是ILC可能因為正電區被疏水結構所遮蔽，因此無法有效包覆ODN。雖然如此，螢光顯微鏡顯示以二元體形式之ILC及CIL均有助於提高細胞攝取效率，皆有明顯的螢光亮點。雷射共軛焦顯微鏡顯示，三種胜肽均可能透過胞吞的方式使細胞攝取複合物，而CIL能助於ODN自內體逃脫而進入細胞質。抑制蛋白質實驗中，藉由ELISA分析被脂多糖(Lipopolysaccharide,LPS)刺激的Raw264.7細胞所表現TNF-α量，結果顯示CIL作為基因載體有較佳的抑制結果，且可維持長時間的藥效。因此可作為具有潛力的去氧寡核苷酸載體。;Indolicidin (IL) is a potential cell-penetrating peptide (CPP), however, its high cytotoxicity restricts its clinical application. In this study, we prepared dimeric IL peptides to promote their biocompatibility and investigate their oligodeoxynucleotide (ODN) delivery efficiencies. Cysteine residues were added to the C or N terminals of IL, by which peptides can be linked as dimers through disulfide bond formation, and we denoted them as ILC and CIL, respectively. HPLC and MASS analyses suggested dimers were successfully prepared in both designs. These dimeric peptides can complex with ODN to self-assembly as nanopartiocles. The MTT results suggested that both ILC and CIL exhibited low cytotoxicity compared to the IL group. The DLS results demonstrated that the sizes of formed peptideplex ranged from 500 to 2000 nm, which were suitable for macrophage uptake. Zeta potentials of peptideplex increased with increasing N/P ratio so that these complexes can be positive charged, however, the absorbance of ILC to ODN is inefficient that the surface charges were always negative except N/P 30. The ODN encapsulation efficiencies of peptides were evaluated by gel retardation assay. Both IL and CIL have good complexation ability to load ODN as stable nanocomplex. In contrast, ILC did not stably complex ODN, which probably correlative to its structure. Because the cationic domain of dimeric ILC is in the middle regions, the hydrophobic domains in two ends probably may mask the positive charges and inhibit the electrostatic interaction between peptide and ODN. However, the fluorescent labeling experiment suggested that both ILC and CIL can promote ODN internalization to Raw264.7 cells. We further investigate the internalization pathway by confocal microscopy, and the results suggested ODN delivered in all groups can be internalized through endocytosis. The long-time observation results indicated that the ODN delivered by CIL can escape from endosome to enter cytosol. Finally, the inhibition experiments were performed against the expression of TNF-α. Anti-TNF-α ODN was delivered to Raw264.7 cell by peptide carriers, and the enzyme-linked immunosorbent assay (ELISA) was performed to measure the expression of TNF-α from Raw264.7 stimulated by lipopolysaccharide (LPS). The results demonstrated CIL demonstrated the best silence against TNF-α, and the inhibition duration can be elongated, suggesting CIL is a potential vector for ODN delivery.
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