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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/80346


    Title: 硬脂基化的Indolicidin作為傳送質體去氧核 酸的非病毒載體;Stearylated Indolicidin as a nonviral vector for plasmid DNA delivery
    Authors: 沈筱容;Shen, Hsiao-Jung
    Contributors: 化學工程與材料工程學系
    Keywords: 硬脂基化;細胞穿膜胜肽;基因傳送;質體DNA;非病毒載體
    Date: 2019-08-20
    Issue Date: 2019-09-03 12:35:30 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 細胞穿透肽(CPP)已經被廣泛研究作為用於基因傳送的非病毒載體。 Indolicidin(IL)是一種有潛力的細胞穿透肽,其富含陽離子精氨酸、賴氨酸和疏水性的色氨酸。雖然IL已被用作小分子核苷酸的載體,例如siRNA和寡脫氧核苷酸(ODN)的傳送,但它不能單獨遞送大分子質體DNA(pDNA)。雙親性分子其親疏水段鍊可自組裝成膠束或脂質體等微結構,藉此與細胞膜融合達到藥物輸送的效果。因此,本研究將IL的N或C末端進行硬脂基化,並分別將命名為sIL和ILs。
    DLS分析硬脂基化胜肽的粒徑及電位,證實sIL和ILs都可進行自組裝,推測結構分別為多層囊泡及反膠束的形式。和DNA複合後分析其裝載率,證實IL的硬脂基化對其與DNA複合的影響不大,而競爭實驗結果顯示自組裝的微結構可以增加複合物的穩定性。由於胜肽載體的輸送取決於其膜擾動的能力,所以我們以鈣黃綠素滲漏實驗進行膜擾動的評估,可以發現N端硬脂化的sIL不利於與膜作用,相較之下,ILs的膜擾動能力與IL相近。當這些胜肽直接用於遞送質粒DNA,從雷射共聚焦顯微鏡和流式細胞儀的結果來看,只有ILs可以促進細胞攝取。轉染結果表明ILs成功轉染HEK-293T細胞,而sIL和IL幾乎沒有轉染。推測sIL的擾膜能力低,且其與DNA的複合物過大,因此不能被宿主細胞攝取。雖然IL可以直接與DNA複合形成合適的大小並且具有優異的包覆率,但穩定性不佳,因此推斷轉染時IL本身易與細胞膜直接吸附而導致DNA在胞外被釋放。相較之下,ILs與DNA複合時,其硬脂基不但可誘使胜肽以其N端與細胞膜接觸達到膜擾動,其自組裝的反膠束結構可增加複合物的穩定性,因此達到促進DNA輸送的目的。
    ;Cell-penetrating peptides (CPP) have been investigated as a non-viral vector for gene delivery. Indolicidin (IL) is a potential cell-penetrating peptide rich in cationic arginine/lysine and hydrophobic tryptophan. Although IL has been applied as a carrier for small nucleotides such as siRNA and oligodeoxynucleotide, it cannot solely deliver huge plasmid DNA (pDNA). Amphiphilic molecules can self-assemble as micelles or liposomes due to their hydrophilic/hydrophobic domains to promote their fusion with cell membranes for drug delivery. Therefore, we modified IL by stearylating its N and C terminals, and denoted them as sIL and ILs, respectively. The DLS analysis was applied to examine the size and surface charges of these stearylated peptides, suggesting that sIL and ILs were capable of self-assembling as multilamellar vesicles (MLVs) and inverted micelles, respectively. The DNA loading examination demonstrated that stearylation of peptides did not hinder their complexation with DNA. The competition experiments showed that self-assembled structure of stearylated peptides increased the stability of complexes. Because delivery efficiencies of CPPs highly depend on their interaction with cell membranes, calcein leakage was applied to evaluation their membrane perturbation ability. In contrast to sIL which demonstrated poor leakage due to its N terminal-stearylation, membrane perturbation ability of ILs was similr to that of unmodified IL. When these peptides were applied for gene delivery, the results of confocal microscopy and flow cytometry showed that only ILs promoted DNA internalization. The transfection results indicated that ILs successfully transfected HEK-293T cells, whereas sIL and IL demonstrated almost no transfection. Due to poor membrane perturbation ability and huge sizes of sIL/DNA complexes, sIL cannot promote DNA transportation. Although IL can directly complex with DNA with appropriate size and good loading efficiency, these complexes is not stable enough. Therefore, IL may adsorb to cell membrane during transfection to release DNA extracellularly. In contrast, ILs may force its N terminus to interact with cell membrane to promote perturbation. The self-assembly of inverted micelles may also stabilize ILs/DNA complexes. These properties explain the improvement effects of ILs on DNA delivery efficiency.
    Appears in Collections:[化學工程與材料工程研究所] 博碩士論文

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