基因治療被視為治療遺傳性疾病、癌症及其他重大疾病的重要策略，而非病毒基因載體的設計為其發展的重要課題。相較於病毒載體，非病毒載體具有免疫原性較低、安全性較高及製備容易等優點，因此受到廣泛關注。酸鹼度敏感型雙子型界面活性劑因具有可質子化官能基，被認為可能促進核內體逃脫，因此本研究合成三種具有不同頭基結構及可質子化官能基數量之酸鹼度敏感型雙子型陽離子界面活性劑，包括12-7NGK-12、12-7NH-12及12-6-12，並利用pH滴定、CMC測定其理化特性、以UV-Vis及DLS分析其DNA凝聚能力，同時進行細胞轉染及細胞毒性評估，以探討可質子化官能基數量對載體性質及基因遞送能力之影響。研究結果顯示，具有較多質子化位點的12-7NGK-12呈現兩個質子化區域、最低的CMC，並可於較低的N/P ratio（載體與DNA之莫耳比）下有效凝聚DNA。DLS顯示三種界面活性劑所形成之複合體粒徑整體上相近，不同界面活性劑對複合物粒徑的影響可以忽略，當複合物在接近電荷中和時會形成較大的聚集體，隨N/P ratio提高會重新穩定並形成適合細胞內吞的奈米複合物。細胞毒性結果亦顯示，12-7NGK-12具有最佳的細胞相容性。綜上所論，雙子型陽離子界面活性劑頭基中可質子化官能基的數量會影響其酸鹼響應特性、DNA凝聚能力及後續基因遞送表現，增加可質子化官能基有助於提升複合體形成能力與細胞相容性，其中12-7NGK-12展現最佳的整體性能。本研究結果有助於釐清頭基結構與載體性質間的關聯，並可作為未來酸鹼度敏感型非病毒基因載體設計之參考。;Gene therapy has emerged as a promising approach for treating genetic disorders， cancer， and other diseases. Non-viral gene carriers have increasingly attracted attention due to their low immunogenicity， improved safety， and ease of preparation. In this study， three pH-responsive cationic gemini surfactants with different head-group structures and numbers of protonatable functional groups (12-7NGK-12， 12-7NH-12， and 12-6-12) were synthesized to investigate the influence of protonatable groups on carrier properties and gene delivery performance. Physicochemical properties were characterized by pH titration and CMC measurements， while DNA condensation and complex size were evaluated by UV–Vis spectroscopy and dynamic light scattering (DLS)， respectively. Transfection efficiency and cytotoxicity were further assessed with cell culture. Among the three surfactants， 12-7NGK-12 exhibited two protonation regimes， the lowest CMC， and efficient DNA condensation at a lower N/P ratio (molar ratio of vector to DNA). DLS analysis showed that the three surfactants formed complexes with similar particle sizes， suggesting a negligible effect of surfactant type on complex size. Near charge neutralization， larger aggregates were formed， while increasing the N/P ratio restabilized the complexes into nanosized particles suitable for cellular uptake. In cellular experiments， 12-7NGK-12 exhibited the highest transfection efficiency and best cytocompatibility. Overall， the results demonstrate that the number of protonatable functional groups influences pH responsiveness， DNA condensation， complex formation， and subsequent gene delivery performance. Among the tested surfactants， 12-7NGK-12 showed the best overall performance， providing insights for the design of pH-responsive non-viral gene delivery systems.
