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Complete Physicochemical Characterization of DNA/Chitosan Complexes by Multiple Detection Using Asymmetrical Flow Field-Flow Fractionation

机译:DNA /壳聚糖复合物的完整理化特性,通过使用不对称流场-流分馏进行多次检测

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摘要

Asymmetrical flow field-flow fractionation (AF4) couplednwith UV-vis spectrophotometry, multiangle light scatteringn(MALS), and dynamic light scattering (DLS)ndetection was used to analyze dispersions of DNArhodamine B labeled chitosan (Ch-rho) complexesnfrequently used as gene delivery vectors. The methodnyielded, in a single experiment, important characteristicsnof the complexes, such as their hydrodynamicnradius, size distribution, conformation, composition,nand the amount of free Ch-rho in the dispersions.nSamples for analysis were obtained by varying experimentalnparameters known to influence the transfectionnefficiency of DNA/chitosan complexes, including thenDNA concentration at mixing (82-164 μg/mL), thenratio of chitosan amino groups to DNA phosphatengroups (3 e N/P ratio e 15), the chitosan molecularnweight (10-76 kDa), and its degree of deacetylation.nIn all preparations, DNA/Ch-rho complexes had hydrodynamicnradii ranging from 15 to 160 nm. Both thenDNA concentration and the Ch-rho molecular weightninfluence the size distribution of the complexes: angreater fraction of large particles was detected inndispersions prepared with the most concentrated DNAnsolution or the Ch-rho of highest molar mass. Allndispersions contained free Ch-rho in solution. The freenCh-rho content ranged from 53 to 92% of the total Chrhonconcentration in dispersions prepared with N/Pnratios from 3 to 15, respectively, implying that the N/Pnratio of the complexes ranged from 1.3 to 1.6 in allnsamples. The accuracy of the free Ch-rho determinationnby AF4/UV-vis/MALS+DLS was confirmed by an independentnmethod involving (1) ultracentrifugation ofnthe dispersions prepared with unlabeled chitosan andn(2) analysis of the supernatant by the Orange II dyendepletion method. This study demonstrates the abilitynof AF4/UV-vis/MALS+DLS to provide a completenphysicochemical characterization of DNA/polycationncomplexes used in nonviral gene delivery.
机译:紫外-可见分光光度法,多角度光散射法(MALS)和动态光散射法(DLS)n检测联用了非对称流场流分馏(AF4),用于分析DNA /去甲胺B标记的壳聚糖(Ch-rho)络合物的分散体。基因传递载体。该方法在单个实验中没有发现复合物的重要特征,例如其流体动力学半径,尺寸分布,构象,组成以及分散液中游离Ch-rho的数量。通过改变已知影响转染效率的实验参数获得分析样品。 DNA /壳聚糖复合物的含量,包括混合时的DNA浓度(82-164μg/ mL),壳聚糖氨基与DNA磷酸根基团的比例(3 e N / P比e 15),壳聚糖分子量(10-76 kDa)和在所有制剂中,DNA / Ch-rho复合物的流体力学半径为15至160 nm。然后DNA浓度和Ch-rho分子量都影响复合物的大小分布:在使用最浓缩的DNAnsolution或最高摩尔质量的Ch-rho制备的分散液中检测到大颗粒的Angerter分数。 Alln分散体在溶液中含有游离的Ch-rho。用N / Pnratios制备的分散液中的freenCh-rho含量范围为总Chrhon浓度的53%至92%,这意味着在Alln样品中,复合物的N / Pnatio范围为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以证实,该方法涉及(1)将未标记壳聚糖制备的分散体超速离心,以及(2)通过Orange II染料耗竭法对上清液进行分析。这项研究表明AF4 / UV-vis / MALS + DLS能够提供用于非病毒基因递送的DNA /聚阳离子复合物的完整物理化学特征。

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  • 来源
    《Analytical Chemistry》 |2010年第23期|p.9636-9643|共8页
  • 作者单位

    Department of Chemical Engineering and Institute of Biomedical Engineering, Ecole Polytechnique de Montre´ al,P. O. 6079 Succursale Centre-Ville, Montreal, Quebec, Canada H3C 3A7, and Department of Chemistry and Facultyof Pharmacy, Universite´ de Montre´ al, P. O. 6128 Succursale Centre-Ville, Montre´ al, Que´bec, Canada H3C 3J7;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 入库时间 2022-08-17 13:36:49

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