Lee Jung Im, Ha Kwon-Soo, Yoo Hyuk Sang
Department of Biomaterials Engineering, School of Bioscience and Bioengineering, Kangwon National University, Chuncheon, Republic of Korea.
Acta Biomater. 2008 Jul;4(4):791-8. doi: 10.1016/j.actbio.2008.01.010. Epub 2008 Feb 2.
Fluorescence resonance energy transfer (FRET) was employed to monitor the molecular dissociation of a chitosan/DNA complex with different molecular weights of chitosan. Chitosan with different molecular weights was complexed with plasmid DNA and the complex formation was monitored using dynamic light scattering and a gel retardation assay. As the chitosan molecular weight increased, a more condensed complex was prepared at various ratios of chitosan to DNA. Plasmid DNA and chitosan were separately labeled with quantum dots and Texas red, respectively, and the dissociation of the complex was subsequently monitored using confocal microscopy and fluorescence spectroscopy. As the chitosan molecular weight in the chitosan/DNA complex increased, the Texas red-labeled chitosan gradually lost FRET-induced fluorescence light when HEK293 cells incubated with chitosan/DNA complex were examined with confocal microscopy. This suggests that the dissociation of the chitosan/DNA complex was more significant in the high molecular weight chitosan/DNA complex. Fluorescence spectroscopy also monitored the molecular dissociation of the chitosan/DNA complex at pH 7.4 and pH 5.0 and confirmed that the dissociation occurred in acidic environments. This finding suggests that the high molecular weight chitosan/DNA complex could easily be dissociated in lysosomes compared to a low molecular weight complex. Furthermore, the high molecular weight chitosan/DNA complex showed superior transfection efficiency in relation to the low molecular weight complex. Therefore, it could be concluded that the dissociation of the chitosan/DNA complex is a critical event in obtaining the high transfection efficiency of the gene carrier/DNA complex.
采用荧光共振能量转移（FRET）技术监测不同分子量壳聚糖与DNA复合物的分子解离情况。将不同分子量的壳聚糖与质粒DNA复合，并通过动态光散射和凝胶阻滞试验监测复合物的形成。随着壳聚糖分子量的增加，在壳聚糖与DNA的不同比例下制备出了更致密的复合物。分别用量子点和德克萨斯红对质粒DNA和壳聚糖进行标记，随后利用共聚焦显微镜和荧光光谱监测复合物的解离情况。当用共聚焦显微镜检查与壳聚糖/DNA复合物孵育的HEK293细胞时，随着壳聚糖/DNA复合物中壳聚糖分子量的增加，德克萨斯红标记的壳聚糖逐渐失去FRET诱导的荧光。这表明壳聚糖/DNA复合物在高分子量壳聚糖/DNA复合物中的解离更为显著。荧光光谱还监测了壳聚糖/DNA复合物在pH 7.4和pH 5.0时的分子解离情况，并证实解离发生在酸性环境中。这一发现表明，与低分子量复合物相比，高分子量壳聚糖/DNA复合物在溶酶体中更容易解离。此外，高分子量壳聚糖/DNA复合物相对于低分子量复合物表现出更高的转染效率。因此，可以得出结论，壳聚糖/DNA复合物的解离是基因载体/DNA复合物获得高转染效率的关键事件。
