利用Indolicidin衍生胜肽促進去氧寡核苷酸輸送;The Use of Indolicidin-derived Peptides for Oligodeoxynucleotide Delivery

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Title:	利用Indolicidin衍生胜肽促進去氧寡核苷酸輸送;The Use of Indolicidin-derived Peptides for Oligodeoxynucleotide Delivery
Authors:	邱茹慧;Chiu,Ju-Hui
Contributors:	化學工程與材料工程學系
Keywords:	聚乙烯亞胺;胜肽;去氧寡核苷酸;polyethylenimine;peptide;oligodeoxynucleotide
Date:	2015-08-26
Issue Date:	2015-09-23 10:55:17 (UTC+8)
Publisher:	國立中央大學
Abstract:	本研究Indolicidin(IL)衍生胜肽對去氧寡核苷酸(Oligodeoxynucleotide, ODN)的輸送效果，其輸送模式可分為兩種:直接將胜肽與ODN座共價接合或是以接枝於分支型聚乙烯亞胺(branched polyethylenimine, bPEI)的方式形成陽離子載體。在直接結合的部份，所形成的胜肽-ODN接合物均具有良好的生物適合性，但是只有ILC、CIL、及R57F89C的接合物可以被送入細胞。而在胜肽接枝bPEI的載體研究中，藉由調控胜肽改質之bPEI與ODN的氮磷(N/P)比，使ODN能以靜電吸引力與胜肽改質的bPEI自組裝成複合粒子。當低N/P比時，負電的ODN吸附在聚陽離子上，因此粒子表面電位為負。粒子大小皆小於600nm，為胞吞作用可進行範圍。和未改質的bPEI相比，經由接枝IL衍生胜肽改質的載體可以提升生物相容性。螢光顯微鏡和流式細胞儀結果顯示，PEI-ILC和PEI-CIL可以促進細胞攝取ODN。雷射共軛焦顯微鏡結果顯示，PEI-ILC和PEI-CIL可以更進一步的使ODN進入細胞質。最後我們進行TNF-α的抑制實驗，結果顯示陽離子型高分子載體反而會刺激RAW264.7細胞產生TNF-α，而無法成功的使基因沉默，只有使用PEI-ILC以低的N/P比攜帶基因，可以明顯降低TNF-α的表達。綜合以上結果，我們認為PEI-ILC不只可以避免刺激RAW264.7細胞，並且可以使基因沉默，此載體有潛力作為基因治療應用。;Indolicidin (IL)-derived peptides were studied of their effects on oligodeoxynucleotide (ODN) delivery. These peptides were either directly conjugated to (ODN) or grafted to branched PEI (bPEI) as polycation carriers. The peptide-ODN conjugates demonstrated low cytotoxicities, however, only the conjugates using ILC, CIL, and R57F89C were translocated. For the grafted bPEI method, these vehicles were complexed with (ODN) in different N/P ratios to form self-assembled nanoparticles through electrostatic interaction. Negative zeta potentials of formed nanocomplexes were found when the N/P ratios were low, suggesting anionic ODNs were adsorbed on the surfaces of polycations. The diameters of nanoparticles were smaller than 600 nm which were suitable for endocytosis. Compared to non-modified bPEI, bPEI conjugated to IL-derived peptides demonstrated superior biocompatibility. Fluorescence microscope and flow cytometry results indicated that both PEI-ILC and PEI-CIL promoted ODN internalization. Images captured by confocal microscope revealed that PEI-ILC and PEI-CIL were capable of delivering ODN to cytosol of host cells. Finally, these gene vehicles were applied to inhibit TNF-α expression. Because RAW264.7 cells were sensitive to polycations, the ODN delivered by these vehicles did not result in gene silence. Instead, the levels of expressed TNF-α were highly elicited. Only PEI-ILC in low N/P (N/P 5) significantly reduced TNF-α. These results suggested that PEI-ILC not only avoided to stimulate RAW264.7 cells but also promoted gene silence, which should be a potential vehicle for gene therapy application.
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