本實驗主要利用疊層組裝(layer-by layer assembly)將幾丁聚醣 (chitosan)與質體 DNA 以靜電作用力的方式在基材表面做堆疊。製備好的電雙層以傅立葉轉換紅外線光譜儀與接觸角分析電雙層的化學 與物理性質，並利用紫外光光譜與石英晶體微量天秤來量化基材上的 幾丁聚醣與質體 DNA 之吸附量，藉以討論不同幾丁聚醣分子量及其 水溶液之 pH 值對 DNA 吸附量與釋放量的影響。我們發現分子量 10k Da 幾丁聚醣與質體 DNA 之疊層組裝有較好的基因釋放效果。以此條件之所製備之幾丁聚醣-DNA 基材應用於細胞轉染，以人類胚胎腎臟細胞株(HEK 293T)作為轉染的對象。MTS 測試證實幾丁聚醣並未明顯影響細胞的活性。製備時隨著幾丁聚醣水溶液 pH 值與電雙層層數的增加，對於轉染細胞的轉染基因效果以及表達時間均較好的效果，原因是 pH 6 的製備條件下，會吸附較多的幾丁聚醣分子，提升與 DNA形成複合體進入到細胞內的機會，而較多的層數也可以延長基因轉染的時間長度。此外，相較於 DNA-幾丁聚醣合成的奈米粒子，疊層組裝的轉染可以延長 2~3 天的基因表現。這些結果均顯示疊層組裝可成為基因傳送的有效方法。 Layer-by-layer (LBL) assembly technique was used to immobilize chitosan and plasmid DNA on material surface using electrostatic forces. The chemistry and physical properties of polyelectrolyte multilayer (PEM) was analyzed by FT-IR and contact angle analysis. UV spectrometry and quartz crystal microbalance (QCM ) were applied to quantify the absorbing amounts of DNA and chitosan. Different molecular weights of chitosan and the pH value of chitosan solutions were examined to determine their effects to the DNA adsorption and release profiles. Because 10k Da chitosan had the highest DNA releasing ability, it was used to prepare PEM for transfecting human embryonic kidney 293T cells (HEK 293T cells). MTS assay suggested that chitosan did not reduce the cell viability. Increasing bilayer numbers and pH values of chitosan solutions may enhance cell transfection efficiencies and elongate gene expression. It may be due to that more chitosan molecules adsorbed on substrates during LBL assembly at pH 6 than at pH 4 conditions. More chitosan molecules may result in more complex formation and thus facilitate gene delivery. Furthermore, higher bilayer numbers prolonged gene expression. Compared to the cells transfected by DNA-chitosan nanoparticles, cells in situ transfected on PEM demonstrated an improved and extended gene expression. These results suggested that the LBL method should be a potential strategy to control gene delivery.
