本研究主要目的為設計及合成低細胞毒性之生物可分解性陽離子型高分子載體，並且針對高分子之分子量、緩衝能力、結構與DNA形成複合體之物理化學性質來探討與轉染效率之相關性。由電泳膠片分析、粒徑分析儀、表面電位儀與原子力顯微鏡來分析此複合體在仿生理體液中物理化學性質，並且觀察在限制核酸酵素內切酶和胎牛白蛋白存在下，高分子載體能否有效地保護質體DNA。由酸鹼滴定曲線變化中，來獲得陽離子型高分子載體被質子化緩衝範圍。
新陽離子型非病毒載體聚胺基甲酸酯:合成與分子量之特性
新陽離子型聚胺基甲酸酯LGEA-PU結構中含有帶正電荷官能基團，且合成出四種重量平均分子量，藉此來探討與轉染之間關係。高分子之分子量越大，所形成複合體粒徑和表面電荷更適合於轉染。在轉染結果中，PEI和LGEA-PU系統相較之下，LGEA-PU59具有最好轉染效率。PEG鏈段間隔地分佈在具胺基甲酸酯的主鏈結構中，可以有效地降低這類陽離子型高分子載體之細胞毒性。
陽離子型聚胺基甲酸酯中胺基特性對轉染效率之影響
為了解陽離子型高分子載體結構中真正與DNA形成複合體之主要部份，及高分子結構中特定官能基來影響Polymer/DNA複合體之物理化學性質，進而調控轉染效率。PEG-PU是側鏈含有三級胺，PPA-PU是主鏈和側鏈皆含有三級胺。PEG-PU已經足夠和質體DNA形成奈米複合體，但是與質體DNA所形成作用力是微弱，因為在胎牛白蛋白環境下，PEG-PU/DNA複合體會不穩定，因而釋放出DNA；PPA-PU/DNA複合體不但在胎牛白蛋白存在下相當穩定，且PPA-PU之緩衝範圍有明顯增加，證實了PPA-PU高緩衝能力和作用力也更加穩定，進而增加了其轉染效率。
結構特性與緩衝能力對生物可分解性聚胺基甲酸酯之轉染效率影響
聚胺基甲酸酯結構特徵與DNA所形成複合體之物理化學特性與轉染效率有著相關性。結果顯示了Poly 2和Poly 3能與DNA鍵結並且形成正電荷之複合體，其粒徑大小符合轉染粒徑之需求。Poly 3展現了最好緩衝能力，其轉染效率比Poly 2 和Poly 1還要好。結果證明了高分子結構中胺基官能基與緩衝能力對於DNA釋放是有影響能力。
側鏈結構上三級胺基團對於陽離子型聚胺基甲酸酯基因載體之轉染效率影響
為了解聚胺基甲酸酯側鏈三級胺結構與轉染效率之間相關性，合成一系列的聚胺基甲酸酯(Poly 1-6)。Poly 6具有最佳轉染效率，這些結果支持著聚胺基甲酸酯轉染能力與結構中胺基數目成正比。側鏈三級胺數目能夠明顯改善聚胺基甲酸酯與DNA形成複合體能力、緩衝能力和轉染效率。 The goal of this project is to design and synthesize the biodegradable cationic polymer with low cytotoxicity that can be used as vector for gene delivery. To optimize the relation between transfection efficiency, the characterizations of molecular weight, buffering capacity, the structure of the polyurethane, and complexes formed by the plasmid DNA with polycation. In this assay show the protection effect of polycations to plasmid DNA against endonucleases and bovine albumin by agarose gel band assay. The acid-base titration profile was obtained for polymers resulting in a proton buffering effect within the endosomal/lysosomal compartments of the cell.
New Cationic Polyurethane as Potential Non-Viral Vector: Synthesis and Characterizations of Molecular Weight
To study the characterizations of molecular weight of LGEA-PU on transfection, polymers with four different molecular weights were prepared. In the particle size and zeta-potential assays, it was found that LGEA-PU with higher molecular weight possessed DNA condensing with results required for transfection. The LGEA-PU59/DNA complexes were able to transfect COS-7 cells in vitro with higher transfection efficiency than the PEI and other LGEA-PU systems. The introduction of PEG into the backbone of polyurethane can provide better cytotoxicity profiles, and increased the hydrophilicity and flexibility of the polymer, promoting the susceptibility to hydrolysis.
Relation between Transfection Efficiency and Structure Characterization of Amino Groups in the Cationic Polyurethanes
The aim of the study is that to optimize the structure in the side chain or backbone of the polyurethane (e.g., targeting groups) so that the transfection efficiency and characterizations of particles formed by the plasmid DNA with polycation can be improved. We synthesized novel water-soluble amino acid-based polyurethanes, PEG-PU bearing tertiary amines in the side chain and PPA-PU bearing tertiary amines in the backbone and side chain. Although the PEG-PU and DNA was able to form complexes, the interpolyelectrolyte force between PEG-PU and DNA was poor, not stable to bovine albumin. It was found that bovine albumin is unable to release any DNA from the PPA-PU/DNA complexes. That PPA-PU has stronger electrostatic force between polymer and DNA and higher pH buffering abilities.
Structural Characterization and Buffering Capacity in Relation to the Transfection Efficiency of Biodegradable Polyurethane
The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than Poly 2 and Poly 1. This study reveals that the amine groups in polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery.
Effect of Side Chain Bearing Tertiary Amino Group on Transfection Efficiencies of Cationic Polyurethanes as Gene Vectors
To explore the side chain bearing tertiary amino groups-transfection efficiency relationships of the polyurethanes, we synthesized a variety of polyurethanes (Poly 1-6). These data showed that Poly 6 demonstrated the most transfection efficiency, and suggested that transfection abilities of cationic polyurethanes were improved by increasing the number of amino unit. It was found that the numbers of side chain bearing tertiary amino groups can significantly improve the DNA condensing abilities, buffering capacities, and transfection efficiencies of the cationic polyurethanes.
