Y. No significant impact on the viability could be observed after 3 h of incubation. Values represent the mean of n 3 Licochalcone-A custom synthesis experiments +SEM. doi:10.1371/journal.pone.0058604.gFigure 6. siRNA injection using GNOME laser transfection. A: Transfection of AlexaFluor488 labeled siRNA into ZMTH3 cells. 88 of the cells stained positive for the siRNA after transfection with the optimized parameters (0.5 mg/cm2 AuNP, 20 mJ/cm2, 50 mm/s). B: Exemplary images of ZMTH3 cells transfected with AlexaFlour488-siRNA and control cells. Scale bar: 100 mm. doi:10.1371/journal.pone.0058604.gIncubation of ZMTH3 cells with AuNPs with a concentration of 0.5 mg/cm2, corresponding to the optimal concentration for GNOME laser transfection, and a ten-fold higher concentration for 3 h did not show a reduction in viability (Fig. 5b). When the incubation time was increased to 24 h a slight decrease in viability could be observed after 48 h, even though it stayed above 90 . AuNP mediated toxicity is a quite complex field and a large number of conflicting results have been published lately, claiming AuNPs to be toxic [33] or non-toxic [34]. This can be explained by the numerous amounts of parameters impacting the toxicity, as the particle material, size, shape, surface coating and the target cell type. Particles in the size range applied in our study might be taken up into the cells by endocytosis [12,35], yielding possibly toxic effects. However, Connor et al. demonstrated that internalized particles are non-toxic [34] and Baumgart et al. observed only minor impairment of the cell viability after incubation with comparably high concentrations of 100 nm gold spheres [12]. Therefore, we CAL 120 suppose the AuNPs under the applied conditions as non-toxic. Very small particles in the size range of 1-2 nm might intercalate the DNA and can lead to genetic mutations and longterm effects [36]. As such small fragments could arise from the laser-particle interaction, this needs to be evaluated in future studies.siRNA transfection and knock downApplying the optimized transfection parameters (20 mJ/cm2, 50 mm/s, 0,5 mg/cm2 200 nm AuNP), we transfected an AlexaFluor488 labeled siRNA into ZMTH3 cells to analyze the actual transfection efficiency. Using flow cytometry the efficiency was determined to be 88 (Fig. 6a). To validate the functional knock down, depletion of GFP fluorescence in ZMTH3 cells expressing destabilized EGFP 
was measured 24 h after laser transfection with anti-GFP siRNA (GFP22 siRNA, [37]). The knock down was measured in terms of fluorescence loss compared to an untreated control. To account for cell losses, fluorescence values were additionally corrected according to the corresponding viability as described above. A decrease in fluorescence of up to 82 was achieved (Fig. 7a). Control cells treated with AuNP and siRNA, but not with the laser only showedGold Nanoparticle Mediated Laser TransfectionTable 1. Shift of the resonance absorbance peak in the absorbance spectra of AuNP after laser irradiation.Sample [mJ/cm2] control 12 20 28 36Peakshift [nm] 0.0 212.6 215.4 219.3 228.3 240.6 60.7 60.2 
60.4 61.0 61.3 61.Values represent the mean of n = 3 experiments 6SEM. doi:10.1371/journal.pone.0058604.ta minor knock down (19 ) for the highest siRNA concentration (100 nM) which is probably linked to the endocytotic uptake of AuNPs. No loss in fluorescence was observed for samples only treated with the laser and siRNA, but without AuNP. To further validate the achieved knock down w.Y. No significant impact on the viability could be observed after 3 h of incubation. Values represent the mean of n 3 experiments +SEM. doi:10.1371/journal.pone.0058604.gFigure 6. siRNA injection using GNOME laser transfection. A: Transfection of AlexaFluor488 labeled siRNA into ZMTH3 cells. 88 of the cells stained positive for the siRNA after transfection with the optimized parameters (0.5 mg/cm2 AuNP, 20 mJ/cm2, 50 mm/s). B: Exemplary images of ZMTH3 cells transfected with AlexaFlour488-siRNA and control cells. Scale bar: 100 mm. doi:10.1371/journal.pone.0058604.gIncubation of ZMTH3 cells with AuNPs with a concentration of 0.5 mg/cm2, corresponding to the optimal concentration for GNOME laser transfection, and a ten-fold higher concentration for 3 h did not show a reduction in viability (Fig. 5b). When the incubation time was increased to 24 h a slight decrease in viability could be observed after 48 h, even though it stayed above 90 . AuNP mediated toxicity is a quite complex field and a large number of conflicting results have been published lately, claiming AuNPs to be toxic [33] or non-toxic [34]. This can be explained by the numerous amounts of parameters impacting the toxicity, as the particle material, size, shape, surface coating and the target cell type. Particles in the size range applied in our study might be taken up into the cells by endocytosis [12,35], yielding possibly toxic effects. However, Connor et al. demonstrated that internalized particles are non-toxic [34] and Baumgart et al. observed only minor impairment of the cell viability after incubation with comparably high concentrations of 100 nm gold spheres [12]. Therefore, we suppose the AuNPs under the applied conditions as non-toxic. Very small particles in the size range of 1-2 nm might intercalate the DNA and can lead to genetic mutations and longterm effects [36]. As such small fragments could arise from the laser-particle interaction, this needs to be evaluated in future studies.siRNA transfection and knock downApplying the optimized transfection parameters (20 mJ/cm2, 50 mm/s, 0,5 mg/cm2 200 nm AuNP), we transfected an AlexaFluor488 labeled siRNA into ZMTH3 cells to analyze the actual transfection efficiency. Using flow cytometry the efficiency was determined to be 88 (Fig. 6a). To validate the functional knock down, depletion of GFP fluorescence in ZMTH3 cells expressing destabilized EGFP was measured 24 h after laser transfection with anti-GFP siRNA (GFP22 siRNA, [37]). The knock down was measured in terms of fluorescence loss compared to an untreated control. To account for cell losses, fluorescence values were additionally corrected according to the corresponding viability as described above. A decrease in fluorescence of up to 82 was achieved (Fig. 7a). Control cells treated with AuNP and siRNA, but not with the laser only showedGold Nanoparticle Mediated Laser TransfectionTable 1. Shift of the resonance absorbance peak in the absorbance spectra of AuNP after laser irradiation.Sample [mJ/cm2] control 12 20 28 36Peakshift [nm] 0.0 212.6 215.4 219.3 228.3 240.6 60.7 60.2 60.4 61.0 61.3 61.Values represent the mean of n = 3 experiments 6SEM. doi:10.1371/journal.pone.0058604.ta minor knock down (19 ) for the highest siRNA concentration (100 nM) which is probably linked to the endocytotic uptake of AuNPs. No loss in fluorescence was observed for samples only treated with the laser and siRNA, but without AuNP. To further validate the achieved knock down w.
