Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity.
Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity.
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DOI：
10.1186/1477-3155-9-47
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发表时间：
2011-10-20
影响因子：
10.2
通讯作者：
Murua Escobar H
中科院分区：
文献类型：
--
作者：
Durán MC;Willenbrock S;Barchanski A;Müller JM;Maiolini A;Soller JT;Barcikowski S;Nolte I;Feige K;Murua Escobar H
当转染外源DNA时，高转染率和低方法诱导的细胞毒性副作用对于达到所需的靶细胞效果是必不可少的。正在评估不同的方法和修改，例如使用纳米颗粒（NPs）来提高转染效率。一些研究集中在np介导方法后获得的转染效率上。然而，比较这些新方法与传统方法的毒性的数据仍然很少。采用不同的np介导和常规转染方法，对转染效率和方法诱导的细胞毒性进行了分析。使用两种真核dna表达质粒转染哺乳动物细胞系MTH53A，采用六种不同的转染方案：常规转染试剂（FuGENE HD， FHD）， FHD与两种不同大小的无稳定剂激光产生的AuNPs (PLAL-AuNPs_S1,_S2)， FHD和市购的AuNPs (Plano-AuNP)，以及两种磁性转染方案。采用荧光显微镜和基于gfp的流式细胞术分析转染后24 h各方案的效率。通过测定细胞增殖和碘化丙啶阳性细胞的百分比（PI%）来评估毒性。免疫荧光法检测重组蛋白的表达情况。在转染方案中加入AuNPs显著提高了pires - hrgfpii - il -12转染的效率（FHD: 16%； AuNPs平均值：28%），而磁体辅助方案没有提高效率。无配体PLAL-AuNPs没有明显的细胞毒性作用，而配体稳定的Plano-AuNPs诱导PI%显著增加，细胞增殖能力降低。对于pres - hrgfpii - rhmgb1转染，PLAL-AuNPs的转染效率明显更高（FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%），而磁体辅助转染的效率明显低于FHD方案。PLAL-AuNPs_S1和_S2的PI%明显较高，但未观察到这些NPs对细胞增殖的一致影响。磁辅助方案效果最差，但确实导致最低的细胞毒性作用。本研究表明，添加AuNPs可显著提高dna表达质粒的转染效率。在一些组合中，各自的细胞毒性根据应用的AuNPs的类型和转染的DNA结构而增加。因此，我们的研究结果表明，对于这些AuNPs的常规使用，必须考虑特定的纳米颗粒配方和DNA结构组合。
Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect. This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.
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影响因子：
14.9
作者：
Kamau SW;Hassa PO;Steitz B;Petri-Fink A;Hofmann H;Hofmann-Amtenbrink M;von Rechenberg B;Hottiger MO
通讯作者：
Hottiger MO
影响因子：
10.2
作者：
Petersen, Svea;Soller, Jan T.;Escobar, Hugo Murua
通讯作者：
Escobar, Hugo Murua
影响因子：
3.9
作者：
Shukla, R;Bansal, V;Sastry, M
通讯作者：
Sastry, M
影响因子：
46.9
作者：
Luo, D;Saltzman, WM
通讯作者：
Saltzman, WM
影响因子：
13.3
作者：
Pan, Yu;Neuss, Sabine;Jahnen-Dechent, Willi
通讯作者：
Jahnen-Dechent, Willi