超音波輔佐幾丁聚糖/海藻酸鹽奈米粒子作為基因轉殖載體之特性研究

Abstract

近年有許多的研究指出超音波能和一些奈米粒子作用，在細胞實驗或體內實驗皆有增加藥物傳遞和基因轉殖效率的效果。這些被用來以超音波輔助的奈米粒子目前已經開始被廣泛研究的包括perfluorocarbon nanoparticles, liposomes, cationic polymers (micelles, PEI, DOTMA etc.) 以及 microbubbles等。在這裡，我們首先以超音波輔助幾丁聚糖奈米粒子，作為基因轉殖的載體，並研究評估其對於基因轉殖效率的影響。我們藉由幾丁聚糖分子和遺傳物質去氧核醣核酸之間電荷相吸的作用，製備出Chitosan-DNA 和Chitosan-alginate-DNA奈米粒子。在各種N/P比例下，Chitosan-DNA奈米粒子的大小約在直徑227–292奈米的範圍內；而Chitosan-alginate-DNA其奈米粒子的大小則落在直徑536–654奈米的範圍。這些以幾丁聚糖為基本組成的粒子可以保護去氧核醣核酸，避免其受到週遭環境中酵素作用的破壞，提供了更有利於基因傳遞的條件。在人類腎臟胚胎細胞實驗中，以超音波輔佐Chitosan-DNA以及Chitosan-alginate-DNA奈米粒子可以分別提升30%和25%的轉殖效率。在人類子宮頸癌細胞Hela的實驗中，我們的Chitosan-DNA奈米粒子表現出不亞於市售基因轉殖產品Lipofectamine™2000的基因轉殖效率。簡言之，超音波和幾丁聚糖奈米粒子皆為當前最有潛力的基因轉殖系統之一，從我們的研究結果中可知，結合兩者，以超音波輔助幾丁聚糖奈米粒子，可以有效的增加基因轉殖的效率，並維持相對較低的細胞殺傷力。

Recently, several studies demonstrated that interaction of ultrasound with nanoparticles; perfluorocarbon nanoparticles, liposomes, cationic polymers (micelles, PEI, DOTMA etc.) and microbubbles, may enhance drug and gene delivery in vitro and in vivo. Here, we first introduced ultrasound to a chitosan-mediated gene delivery system, and investigated its effect on the transfection efficiency of this system. During ionic gelation, chitosan and pAcGFP1-C1 plasmid DNA spontaneously interacted to yield Chitosan-DNA nanoparticles (Chi-DNA NPs) with mean Z-average diameters range of 227–292 nm while Chitosan-alginate-DNA nanoparticles (Chi-alg-DNA NPs) had diameters range of 536–654 nm at various N:P ratio, which could potentially protect DNA from nuclease attack and provide beneficial properties for gene delivery. In 293T cells, the transfection efficiency could be enhanced by ultrasound up-to 30% in Chi-DNA NPs (at 3:1 & 5:1 N:P ratio) and 25% improvement of total transfected cells in Chi-alg-DNA NPs (at all ratio we tested). In comparison, transfection efficiency in Hela cells of our system was similar to Lipofectamine™2000. In sum, the combination of the two promising gene transfection strategies, chitosan-based nanoparticle and ultrasound-induced gene delivery system, could enhance gene transfection in vitro depending on the cell type with relatively minor cytotoxicity compared to the commercial liposome gene delivery agent.