Department of Chemical Engineering and Institute of Biomedical Engineering, Ecole Polytechnique de Montréal, P. O. 6079 Succursale Centre-Ville, Montreal, Quebec, Canada H3C 3A7.
Anal Chem. 2010 Dec 1;82(23):9636-43. doi: 10.1021/ac100711j. Epub 2010 Nov 5.
Asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis spectrophotometry, multiangle light scattering (MALS), and dynamic light scattering (DLS) detection was used to analyze dispersions of DNA/rhodamine B labeled chitosan (Ch-rho) complexes frequently used as gene delivery vectors. The method yielded, in a single experiment, important characteristics of the complexes, such as their hydrodynamic radius, size distribution, conformation, composition, and the amount of free Ch-rho in the dispersions. Samples for analysis were obtained by varying experimental parameters known to influence the transfection efficiency of DNA/chitosan complexes, including the DNA concentration at mixing (82-164 μg/mL), the ratio of chitosan amino groups to DNA phosphate groups (3 ≤ N/P ratio ≤ 15), the chitosan molecular weight (10-76 kDa), and its degree of deacetylation. In all preparations, DNA/Ch-rho complexes had hydrodynamic radii ranging from 15 to 160 nm. Both the DNA concentration and the Ch-rho molecular weight influence the size distribution of the complexes: a greater fraction of large particles was detected in dispersions prepared with the most concentrated DNA solution or the Ch-rho of highest molar mass. All dispersions contained free Ch-rho in solution. The free Ch-rho content ranged from 53 to 92% of the total Ch-rho concentration in dispersions prepared with N/P ratios from 3 to 15, respectively, implying that the N/P ratio of the complexes ranged from 1.3 to 1.6 in all samples. The accuracy of the free Ch-rho determination by AF4/UV-vis/MALS+DLS was confirmed by an independent method involving (1) ultracentrifugation of the dispersions prepared with unlabeled chitosan and (2) analysis of the supernatant by the Orange II dye depletion method. This study demonstrates the ability of AF4/UV-vis/MALS+DLS to provide a complete physicochemical characterization of DNA/polycation complexes used in nonviral gene delivery.
不对称流场流分离（AF4）与紫外-可见分光光度法、多角度光散射（MALS）和动态光散射（DLS）检测联用，用于分析经常用作基因传递载体的 DNA/若丹明 B 标记壳聚糖（Ch-rho）复合物的分散体。该方法在单次实验中提供了复合物的重要特性，例如其水动力半径、粒径分布、构象、组成以及分散体中游离 Ch-rho 的量。通过改变已知影响 DNA/壳聚糖复合物转染效率的实验参数获得用于分析的样品，包括混合时的 DNA 浓度（82-164μg/mL）、壳聚糖氨基与 DNA 磷酸盐基团的摩尔比（3≤N/P 比≤15）、壳聚糖分子量（10-76kDa）及其脱乙酰度。在所有制剂中，DNA/Ch-rho 复合物的水动力半径在 15-160nm 之间。DNA 浓度和 Ch-rho 分子量均影响复合物的粒径分布：在用最浓 DNA 溶液或摩尔质量最高的 Ch-rho 制备的分散体中，检测到更大比例的大颗粒。所有分散体均含有游离的 Ch-rho。在 N/P 比为 3 至 15 的分散体中，游离 Ch-rho 的含量范围分别为分散体中总 Ch-rho 浓度的 53%至 92%，这意味着在所有样品中复合物的 N/P 比范围均为 1.3 至 1.6。通过 AF4/UV-vis/MALS+DLS 测定游离 Ch-rho 的准确性通过一种独立的方法得到了证实，该方法涉及（1）用未标记的壳聚糖制备的分散体的超速离心，以及（2）用橙 II 染料耗竭法分析上清液。本研究表明，AF4/UV-vis/MALS+DLS 能够提供用于非病毒基因传递的 DNA/聚阳离子复合物的完整物理化学特性。
