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Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini–Bolaamphiphilic Hybrid Lipid
ACS Omega ( IF 3.7 ) Pub Date : 2018-01-08 00:00:00 , DOI: 10.1021/acsomega.7b01657 María Martínez-Negro 1 , Andrés Guerrero-Martínez 1 , Luis García-Río 2 , Òscar Domènech 3 , Emilio Aicart 1 , Conchita Tros de Ilarduya 4 , Elena Junquera 1
ACS Omega ( IF 3.7 ) Pub Date : 2018-01-08 00:00:00 , DOI: 10.1021/acsomega.7b01657 María Martínez-Negro 1 , Andrés Guerrero-Martínez 1 , Luis García-Río 2 , Òscar Domènech 3 , Emilio Aicart 1 , Conchita Tros de Ilarduya 4 , Elena Junquera 1
Affiliation
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A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini–bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive (
) and negative charges (
), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100–200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/– charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.
中文翻译：
基于 Gemini-Bola 两性杂合脂质的非病毒纳米载体转染质粒 DNA 的多学科方法
本文提出了一种包括生化和生物物理研究在内的多学科策略，以评估由 Gemini-bola 两亲性脂质 (C 6 C 22 C 6 ) 和众所周知的辅助脂质 1,2-混合物组成的脂质纳米聚集体的潜力。二油酰-sn-甘油-3-磷脂酰乙醇胺 (DOPE) 以高效、安全的方式将质粒 DNA 转染到活细胞中。为此，采用了多种实验技术，例如 zeta 电位（相分析光散射方法）、琼脂糖凝胶电泳（pDNA 压实和 pDNA 保护测定）、小角 X 射线散射、低温透射电子显微镜、原子力显微镜、荧光辅助细胞分选、发光测定和细胞毒性测定。结果显示，阳离子脂质和质粒分别仅提供其标称正电荷 () 和负电荷 ( ) 的 70% 和 30%。与 DOPE 混合后，它们形成脂质复合物，在典型的多层 L α溶致液晶纳米结构中自聚集，尺寸在 100-200 nm 范围内，多分散性低，非常适合保留在血流中并穿过细胞膜。有趣的是，这些纳米聚集体能够压缩、保护（免受 DNase I 的降解作用）并将两种 DNA 质粒（pEGFP-C3，编码绿色荧光蛋白和 pCMV-Luc，编码荧光素酶）转染到 COS-7 细胞中，只要有效+/-电荷比保持高于1但相当接近电中性，其效率等于甚至优于通用控制Lipo2000*。此外，这种转染过程没有细胞毒性，因为 COS-7 细胞的活力保持在高水平，大于 80%。所有这些特征使 C 6 C 22 C 6 /DOPE 纳米系统成为体外最佳的非病毒基因纳米载体，并成为未来体内实验的潜在候选者。
更新日期：2018-01-08
) and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100–200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/– charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.
中文翻译：
基于 Gemini-Bola 两性杂合脂质的非病毒纳米载体转染质粒 DNA 的多学科方法
本文提出了一种包括生化和生物物理研究在内的多学科策略，以评估由 Gemini-bola 两亲性脂质 (C 6 C 22 C 6 ) 和众所周知的辅助脂质 1,2-混合物组成的脂质纳米聚集体的潜力。二油酰-sn-甘油-3-磷脂酰乙醇胺 (DOPE) 以高效、安全的方式将质粒 DNA 转染到活细胞中。为此，采用了多种实验技术，例如 zeta 电位（相分析光散射方法）、琼脂糖凝胶电泳（pDNA 压实和 pDNA 保护测定）、小角 X 射线散射、低温透射电子显微镜、原子力显微镜、荧光辅助细胞分选、发光测定和细胞毒性测定。结果显示，阳离子脂质和质粒分别仅提供其标称正电荷 (
) 和负电荷 ( ) 的 70% 和 30%。与 DOPE 混合后，它们形成脂质复合物，在典型的多层 L α溶致液晶纳米结构中自聚集，尺寸在 100-200 nm 范围内，多分散性低，非常适合保留在血流中并穿过细胞膜。有趣的是，这些纳米聚集体能够压缩、保护（免受 DNase I 的降解作用）并将两种 DNA 质粒（pEGFP-C3，编码绿色荧光蛋白和 pCMV-Luc，编码荧光素酶）转染到 COS-7 细胞中，只要有效+/-电荷比保持高于1但相当接近电中性，其效率等于甚至优于通用控制Lipo2000*。此外，这种转染过程没有细胞毒性，因为 COS-7 细胞的活力保持在高水平，大于 80%。所有这些特征使 C 6 C 22 C 6 /DOPE 纳米系统成为体外最佳的非病毒基因纳米载体，并成为未来体内实验的潜在候选者。
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