Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>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.</p> <p>Results</p> <p>The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-<it>eIL-12 </it>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-<it>rHMGB1 </it>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.</p> <p>Conclusions</p> <p>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.</p

Picosecond Laser Fabrication of Functional Gold–Antibody Nanoconjugates for Biomedical Applications

Conventional fabrication of gold nanoconjugates is often accomplished by multistep chemical synthesis, causing rather long production times (hours to days) and requiring multiple purification steps. In contrast, by applying femtosecond-laser systems the process of pulsed laser ablation in liquids (PLAL) with in situ bioconjugation may be used alternatively to produce surfactant-free and functional nanoconjugates within a single-step approach on the time scale of minutes. Gold nanoconjugates functionalized with nucleic acids, peptides, proteins, and aptamers were successfully established by these means. However, limited process productivity is a main disadvantage of the femtosecond-PLAL approach due to the short pulse duration. In this work for the first time, we utilize picosecond-PLAL to fabricate novel gold–antibody nanoconjugates for cellular staining issues. The functionality of the nanoconjugates is confirmed by blotting and cellular immunolabeling, resulting in equivalent staining results than achieved with conventional labeling markers. By the adoption of picosecond pulse duration a higher productivity by 1 order of magnitude is reached compared to the conventional femtosecond-PLAL. Moreover, the production of nanoparticles and nanoconjugates with the same surface composition, the same amount of biomolecule load and the same level of biomolecule structure integrity is proven than that gained by femtosecond-PLAL. Finally, the potential physical mechanisms of biomolecule degradation and the quantitative online monitoring of the degradation are discussed. The results emphasize laser-fabricated gold–antibody nanoconjugates as competing products to commercial immunoflow or cellular staining markers. Moreover, picosecond-PLAL enables a significantly higher production speed of gold nanobiohybrids than that achieved via existing fabrication methods and therefore represents a competing technology

Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos