Figure 1.
A: Schematic drawing and B: photograph of the setup.
Figure 2.
Experimental procedure for GNOME laser transfection.
Cells are incubated with AuNP (1), the molecule to be delivered is added (2) and the sample is irradiated to permeabelize the cell membrane (3). Drawings are not true to scale.
Figure 3.
Fluorescence level and viability for different transfection parameters for the delivery of 10 kDa FITC-dextran.
A: Fluorescence for different scanning velocities at a constant AuNP concentration of 5 µg/cm2. The scanning velocity was set to values between 10 and 200 mm/s, corresponding to 175 and 9 pulses per point, respectively. For all velocities the same fluorescence level could be achieved by using increasing radiant exposure for higher velocities as indicated by the dotted line. B: The corresponding viability dropped with higher radiant exposure and slower scanning velocities. C, D: High AuNP concentrations led to pronounced cell death even at low values of radiant exposure and thus only provide low levels of fluorescence. Most efficient delivery was achieved at 0.5 µg/cm2 and 20 mJ/cm2 as indicated by the dotted line in C. Scanning velocity was kept constant at 50 mm/s. Data points represent the mean of n = 4 experiments.
Figure 4.
A: Particle count per cell after 3 hours of incubation with different AuNP concentrations. No significant difference in the particle count was observed after irradiation with 20 mJ/cm2 (p = 0.46). Values represent the mean of n = 3 experiments ±SEM. B: ESEM image of one exemplary ZMTH3 cell incubated for 3 h with 0.5 µg/cm2 200 nm AuNP in RMPI. C An individual cell was marked and the particles were counted.
Figure 5.
Course of the viability after GNOME laser transfection and AuNP incubation.
A: The cells have been incubated with 0.5 µg/cm2 200 nm AuNP and were irradiated with different values of radiant exposure. At the indicated time points after laser treatment the cell viability was measured. For all conditions tested the viability stayed above 80%. B: ZMTH3 cells were incubated with 200 nm AuNP for 3 and 24 h respectively. No significant impact on the viability could be observed after 3 h of incubation. Values represent the mean of n≥3 experiments +SEM.
Figure 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 µg/cm2 AuNP, 20 mJ/cm2, 50 mm/s). B: Exemplary images of ZMTH3 cells transfected with AlexaFlour488-siRNA and control cells. Scale bar: 100 µm.
Figure 7.
siRNA mediated GFP knock down.
A: GFP Fluorescence depletion after GNOME transfection of different siRNA concentrations. No pronounced knock down was observed in the laser and AuNP controls. Values represent the mean of n = 3 experiments +SEM. B: Western blot of β-Actin and GFP after siRNA mediated knock down of GFP. The sample shows a 55% reduction in the GFP band intensity.
Figure 8.
Absorbance spectra of 200 nm AuNP in RPMI after irradiation with different values of radiant exposure.
Table 1.
Shift of the resonance absorbance peak in the absorbance spectra of AuNP after laser irradiation.