br Fig Fluorescence microscopy images
Fig. 5. Fluorescence microscopy images of H1299 G-418 Sulfate 48 h after transfection with MDEA/DOPE particles containing siMM or siGFP siRNA and 3.26 μM Nile red. The particles were made using the thin film method and at N:P and D:M ratios of 6 and 3, respectively. (For interpretation of the re-ferences to color in this figure, the reader is referred to the web version of this article.)
Fig. 6. Nile red fluorescence intensity of a Cal27 cell line measured 48 h after transfection with pure Nile red or MDEA/ DOPE particles made with siRNA and Nile red at two different D:M ratios. The particles were made using the thin film method and at a N:P ratio of 6. Shown are average fluores-cence intensity normalized to the non-treated control as measured by flow cytometry (left) and fluorescence micro-scopy images of red Nile red fluorescence for pure Nile red (top right) and particles with Nile red (lower right). Note the logarithmic scale.
fluorescence when placed in different environments (Greenspan and Fowler, 1985). When particles were passed through any of the filters, the filtrate did not contain Nile red fluorescence above the level of detection. This concentration level was determined by a standard curve for Nile red to be a detection limit of 100 ng/mL Nile red (Supple-mentary Fig. 1). With the ultrafiltration columns, we were able to re-suspend the retentate after filtration, and this retentate showed fluor-escence. These experiments strongly indicate that all Nile red is incorporated into the particles and that these samples contain no free Nile red. That free Nile red is more fluorescent than formulated Nile red may explain the observation in Fig. 6 that free Nile red induces greater cellular fluorescence than formulated Nile red.
To investigate the degree to which the Nile red was released from the particles in aqueous environments we performed a dialysis release assay over 7 days. No Nile red fluorescence was observed in the dialysis medium (detection limit of 100 ng/mL, Supplementary Fig. 1) at any time point despite the membranes molecular weight cut-off of 14 kDa (the molecular weight of Nile red is 318 Da). This indicates that the Nile red is strongly bound to the particles.
To investigate whether the MDEA/DOPE particles with siRNA and with or without etoposide are toxic to the cells, resazurin based
viability assays were performed on H1299 and Cal27 cells (Fig. 7) treated with these particles. The viability of H1299 cells 24 h after transfection with MDEA/Dope siRNA particles without etoposide at
Filtration of free Nile red and particles incorporating Nile red (D:M ratio 1, N:P 6), fluorescence was used to quantify Nile red before and after filtration. LOD is level of detection (~0.1 μg/mL).
Sample Method Before [μg/mL] Standard deviation After [μg/mL] Standard deviation
Fig. 7. Normalized viability [%] of H1299 and Cal27 cells 24 h after treatment with MDEA/DOPE siRNA particles at different D:M ratios and with or without etoposide at different concentrations. The particles were made using the thin film method and at a N:P ratio of 6.
Dynamic light scattering of particles made using the thin film method at dif-ferent D:M ratios with and without Nile red and etoposide, the N:P ratio is 6. Table shows intensity averaged diameters and standard deviation of three measurements.
Sample Average diameter (nm) Standard deviation (nm)
at D:M ratios of 1, 2 and 3 and different levels of etoposide yielded a similar result (Supplementary Fig. 2), in this experiment, none of the particle samples without etoposide were toxic compared to the control samples, but all particles with etoposide showed statistically significant toxicity except for those made with a D:M ratio of 1 and a 3.4 μM and 6.8 μM etoposide (the lowest etoposide levels tested).