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  • 學位論文

開發還原及酸鹼敏感性透明質酸─聚乙烯亞胺奈米顆粒包覆抑制血管新生質體作為幹細胞基因治療之研究

Redox and pH Sensitive HA-ss-PEI Nanocomplex Encapsulated Endostatin Plasmid for Stem Cell Gene Therapy

指導教授 : 王子威
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摘要


藉由發展可轉染幹細胞的基因載體,將可使基因治療方向由抑制不正常細胞增生的被動方向,朝向以幹細胞修復受損組織的主動模式發展,對未來臨床治療提供更有效的策略。然而,目前市面上常見的基因載體能夠有效轉染幹細胞的商品有限,且大部分可轉幹細胞的載體生物相容性仍有疑慮。本研究主要開發一種可有效轉染幹細胞的基因傳輸奈米載體,利用天然高分子─透明質酸 (Hyaluronic Acid) 化學改質接枝上具有氧化還原、酸鹼敏感性的官能基 (硫醇─雙硫鍵結),再和聚乙烯亞胺 (Polyethylenimine) 利用化學交聯方式形成透明質酸-聚乙烯亞胺 (HA-ss-PEI) 作為材料。材料帶正電的特性可以與帶負電質體 (pDNA) 形成靜電力複合體,顆粒大小約為100奈米左右,可藉由細胞胞吞作用進入細胞中;內吞作用中外來物多傳輸至核內體 (Endosome),其酸性環境(約pH4-5)及細胞內還原環境使複合體膨漲、崩解,並釋出攜帶的質體。目前對於基因載體的研究十分廣泛,如何提高轉染率及有效降低生物毒性這兩點是目前基因載體發展的兩大方向。研究中使用的透明質酸為構成細胞外基質的成分,能夠促進細胞胞吞作用,雙硫鍵結則由胺基酸組成;在聚乙烯亞胺的部分則選擇小分子的材料,可以有效降低因轉染對細胞造成的傷害,聚乙烯亞胺在核內體可藉由質子海綿效應,搭配環境敏感的雙硫鍵使基因物質有效釋出,同步達成高轉染率及生物相容性的目的。結果顯示本研究開發的透明質酸─聚乙烯亞胺 (HA-ss-PEI) 可有效轉染不易轉染的幹細胞,並且不產生大部分基因載體會使細胞數量減少的問題。除此之外,我們也成功利用此基因載體,將血管生成抑制劑的基因載入間葉幹細胞,使其持續性的製造並釋放抗血管新生因子Endostatin (即血管生成抑制劑),並在培養環境中模擬軟骨再生環境誘導間葉幹細胞朝向透明軟骨分化確認轉染後的幹細胞仍保有可分化的多功能性。由於此基因載體的主材料成分為透明質酸,與目前治療關節炎注射透明質酸的方法使用相同材料,容易為臨床醫生所接受,具有臨床價值。

並列摘要


Gene therapy strategies can be useful for tissue engineering by modifying stem cells directly using various gene delivery carriers. Gene delivery offers a new and potentially promising treatment modality for the inherited genetic diseases or disorders. The development of delivery systems able to alter the biological profiles of therapeutic agents (viz., pDNA, siRNA, growth factors, drugs, etc.) is considered of utmost importance in biomedical research and the pharmaceutical industry. In this study, a redox- and pH-sensitive hyaluronic acid (HA) - polyethylenimine (PEI) copolymer with disulfide linkage was synthesized, characterized and examined as a potential non-viral gene vector. The physical and chemical properties of fabricated gene carrier were analyzed via 1H NMR and FT-IR for the demonstration of crosslinking of HA with PEI. TNBS assay and Ellman’s reagent were conducted for the verification of disulfide bond formation and crosslinking degree. The size, morphology and surface charge of nanoparticle were investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta potential, respectively. The ability of HA-ss-PEI conjugate to complex with plasmid DNA was observed by gel electrophoresis and the optimal N/P ratio was also determined. Fluorescent microscopy and ELISA spectroscopy were utilized to examine transfection efficiency and protein expression level of therapeutic pDNA. Finally, the ability of transfected hMSCs to differentiate into chondrocyte was investigated by Alcian blue stain and immunocytochemistry (ICC) for type II collagen. From the results, a novel redox- and pH-sensitive gene delivery nanocarrier has been successfully synthesized. The positive HA-ss-PEI conjugate complexed with negative charged plasmid DNA can be achieved via electrostatic attraction to form a stable spherical nanoparticle of 100 nm in diameter. N/P=2 possessed the optimal condition for releasing encapsulated plasmid when triggered by stimuli such as redox reaction and pH change. The nanocarrier could successfully complex with pDNA and transfect stem cells to produce specific protein for therapeutic purposes. hMSCs transfected with HA-ss-PEI/pEndo complexes could produce endostatin, an anti-angiogenic agent, and differentiate into chondrocytes after 7 days chondrogenic induction. In brief, the redox- and pH-sensitive HA-ss-PEI nanoparticles can be used as a promising non-viral gene carrier for stem cell gene therapy.

參考文獻


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