"Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide.
"Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide.
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DOI：
10.1007/s11095-009-9976-1
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发表时间：
2009-12
影响因子：
3.7
通讯作者：
Berkland, Cory
中科院分区：
文献类型：
--
作者：
Baoum, Abdulgader;Xie, Sheng-Xue;Fakhari, Amir;Berkland, Cory
通常，低分子量阳离子肽或聚合物由于无法将质粒 DNA 浓缩成小纳米颗粒而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。随着钙浓度的增加，研究了 pDNA 与 TAT 或 PEI 的静电复合物。使用凝胶电泳来测定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。评估复合物内磷酸盐和胺基团的可及性以确定钙对结构的影响。通过优化钙浓度，TAT/pDNA 复合物被浓缩成 50-100 nm 的小颗粒。针对小尺寸优化的复合物在 A549 细胞中也表现出比 PEI 复合物更高的转染效率。 TAT 和 TAT 复合物在浓度高达 5 mg/mL 时表现出可忽略不计的细胞毒性，而 PEI 表现出高细胞毒性，正如预期的那样。探测 TAT-Ca/pDNA 结构表明，钙与磷酸基和胺基相互作用以压实复合物；然而，这些“软”交联可能会被竞争性破坏，以促进 DNA 释放。通过“软”钙交联获得了小且稳定的 TAT-Ca/pDNA 复合物，导致持续的基因表达水平高于对照 PEI 基因载体观察到的水平。 TAT-Ca/pDNA 复合物稳定，即使在存在 10% FBS 的情况下也能保持粒径和转染效率。 TAT-Ca 复合物提供了一种有效的载体，为可翻译基因传递提供了潜力。
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.
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影响因子：
3
作者：
Felgner, PL
通讯作者：
Felgner, PL
影响因子：
4.8
作者：
Derossi, D;Calvet, S;Prochiantz, A
通讯作者：
Prochiantz, A
影响因子：
4.8
作者：
Eguchi, A;Akuta, T;Nakanishi, M
通讯作者：
Nakanishi, M
影响因子：
4.2
作者：
Abdallah, B;Hassan, A;Demeneix, BA
通讯作者：
Demeneix, BA
影响因子：
3.7
作者：
Haberland, A;Knaus, T;Böttger, M
通讯作者：
Böttger, M