陽離子高分子被使用在基因傳遞載體應用時會產生一些不利的副作用，譬如游離陽離子會造成細胞毒性等。藉由移除過多的正電荷希望可以發展成為安全的基因傳遞系統。然而，經由陰離子包覆在陽離子/DNA複合體表面已被報導具有增進DNA複合體的轉染效果及降低其細胞毒性。因此，本研究的目的主要是研究PAMAM/DNA複合體之游離陽離子及陰離子包覆在結構上與細胞毒性上的影響。在本研究中，我們使用離心的方式移除PAMAM/DNA複合體游離的正電荷，而其複合體再包覆上不同的陰電性物質如海藻酸、果膠、玻尿酸、及牛血清白蛋白。其結構特性我們使用粒徑大小、界面電位和細胞毒性分析。PAMAM/DNA複合體藉由包覆帶陰電性的多醣體與牛血清白蛋白會導致粒徑大小增加及界面電位為負電位。包覆果膠與海藻酸於PAMAM/DNA複合體上可以導致Raw264.7細胞的細胞毒性減少，而果膠又比海藻酸更能改善細胞存活率作用。
未移除過多正電荷的PAMAM/DNA複合體的平均粒徑明顯比藉由離心法移除過多正電荷大。PAMAM/DNA複合體的細胞毒性也藉由移除過多正電荷而降低。移除過多正電荷後，PAMAM/DNA複合體藉由包覆陰離子物質如海藻酸、玻尿酸、牛血清白蛋白及果膠，而導致平均粒徑的增加。除了牛血清白蛋白之外，界面電位也因為陰電性的濃度增加而由正電位轉變為負電位。
結果顯示PAMAM/DNA複合體包覆不同具功能性的陰電性物質於以樹狀體為基礎的基因傳遞系統在未來可達到最佳化的發展。 Cationic polymers used for gene delivery vectors were known to induce some severe adverse effects such as cytotoxicity by its excess cationic charge. The removal of free polycations would be desirable for the development of safe gene delivery systems. Also, recharging cationic DNA complexes with anionic coating has been reported to enhance the transfection performance and reduce cytotoxicity of DNA complexes. Therefore, the purpose of this study was to investigate the effect of free polycatons and anionic coating on the structure and cytotoxicity of polyamidoamine (PAMAM)/DNA complexes. In our study, free polycations are removed from the solutions of PAMAM/DNA complexes by filtration, and the resulting complexes were then coated with various anionic substances such as alginate, pectin, hyaluronic acid (HA), and bovine serum albumin (BSA). The structural properties were confirmed using particle size, zeta potential, and cytotoxic assay. Anionic coatings with polysaccharides and BSA resulted in the increases of particle size of PAMAM/DNA complexes and were shown as negative zeta potentials. Coatings with pectin and alginate resulted in the decreases of cytotoxicity in Raw 264.7 cells treated with PAMAM/DNA complexes and pectin had better protective effects on cell viability than alginate.
The average particle size for PAMAM/DNA complexes without removal of excess polycations is significantly larger than that with removal of excess polycations by centrifugation. The cytotoxicity of PAMAM/DNA complexes is reduced with removal of excess polycations. After removing excess polycations, the average particle size for PAMAM/DNA complexes is increased by anionic coating with alginate, hyaluronic acid, bovine serum albumin, and pectin. Except BSA, the zeta potential of the complexes changed from positive values to negative ones by the increases of anionic concentrations.
The results suggested that anionic coating of PAMAM/DNA complexes with various functional materials deserved to be further developed to optimize the performance of dendrimer-based gene delivery systems.
