Department of Pharmaceutical Chemistry, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA.
Pharm Res. 2009 Dec;26(12):2619-29. doi: 10.1007/s11095-009-9976-1. Epub 2009 Sep 30.
Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks.
Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure.
TAT/pDNA complexes were condensed into small, 50-100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these "soft" crosslinks could be competitively disrupted to facilitate DNA release.
Small and stable TAT-Ca/pDNA complexes were obtained via "soft" calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.
通常，由于无法将质粒 DNA 浓缩成小纳米颗粒，低分子量阳离子肽或聚合物表现出较差的转染效率。在这里，使用含有钙交联的 TAT/pDNA 复合物实现了有效的基因传递。
研究了与 TAT 或 PEI 静电结合的 pDNA 与钙离子浓度增加时的复合物。凝胶电泳用于确定 DNA 凝聚。通过透射电子显微镜探测复合物的形态。使用荧光素酶报告质粒评估转染效率。评估了复合物中磷酸和胺基的可及性，以确定钙对结构的影响。
通过优化钙的浓度，TAT/pDNA 复合物被凝聚成 50-100nm 的小颗粒。优化为小尺寸的复合物在 A549 细胞中的转染效率也高于 PEI 多聚物。TAT 和 TAT 复合物在高达 5mg/ml 的浓度下表现出可忽略的细胞毒性，而 PEI 则表现出高细胞毒性，这是预期的。探测 TAT-Ca/pDNA 结构表明，钙与磷酸和胺基相互作用以压缩复合物;然而，这些“软”交联可以被竞争性破坏以促进 DNA 释放。
通过“软”钙交联获得了小而稳定的 TAT-Ca/pDNA 复合物，导致观察到的基因表达水平高于对照的 PEI 基因载体。TAT-Ca/pDNA 复合物稳定，即使在存在 10% FBS 的情况下，也能保持颗粒大小和转染效率。TAT-Ca 复合物提供了一种有效的载体，具有潜在的可转化基因传递能力。
