Figure 1.
CM18-Tat11/DNA plasmid complex characterization.
(a) Hypothetical ionic interaction model between DNA plasmid (brown fragment with blue negative charges) and CM18-Tat11 peptides (grey: CM18 and black: Tat11 with red positive charges). (b) CM18-Tat11 DNA condensation ability analyzed by EtBr exclusion assay. Results for complexes at N:P ratio from 0: 1 to 32: 1 are given as relative fluorescence and a value of 100% is attributed to the fluorescence of naked DNA with EtBr. The reported values represent the mean of three independent measurements, each performed in triplicate. (c) Stability of CM18-Tat11/plasmid DNA binary complex at N/P ratio as in (b) evaluated by agarose gel electrophoresis assay. Complexes are electrophoresed on a 0.8% (w/v) agarose gel with TBE running buffer at 80 V for 40 minutes. (d) ξ-potentials of CM18-Tat11/plasmid DNA binary complexes with N/P ratios as in (a) are measured at 25 °C by a Zetasizer Nano ZS90 instrument equipped with a red laser of wavelength 630 nm. Each sample is observed with 20 repeated measurements across 3 trials. Error bars in figures indicate standard deviations.
Figure 2.
CM18-Tat11/DNA plasmid complex in vitro transfection efficiency and cytotoxicity.
(a) Transgene expression is detected 24 hours after transfection by measuring luciferase activity from an aliquot of the HeLa cells external medium. Light grey column is the mean value obtained with naked DNA, dark grey column is for lipofectamine, while red and black column are respectively for CM18-Tat11 and Tat11 DNA complex at N:P ratio from 4:1 to 32:1. The reported RLU/well values represent the mean of three independent measurements, each performed in triplicate. (b) Wst-8 assay to evaluate cell metabolic activity. The wst-8 reagent was added for 2 hours and absorbance at 450 nm measured. Untreated cells are defined as 100% viable, while cells exposed to 20% dimethyl sulfoxide (white column) are used as positive control for decreased metabolic activity.
Figure 3.
Spectroscopic properties and cell uptake dynamics of CM18-Tat11-atto633/DNA plasmid-Cy3 complexes.
(a, b, c, d) Fluorescence spectra are recorded at 37 °C on a spectrofluorometer by exciting at 540 nm and collecting the fluorescence between 550 and 800 nm. First, a fluorescence emission measurement is performed on a PBS solution of Cy3-labeled DNA plasmid alone (solid green line in a, b, c, d). Then, atto633-labeled (dark yellow line in a) or unlabeled (dashed green line line in b) CM18-Tat11 or atto633-labeled Tat11 (dark yellow line in c) or atto633-labeled CM18 (red line in d) at N:P ratio 16:1 is added, and the emission spectrum recorded again (e) Nomarsky images of CLSM timelapse assay performed on cells incubated with CM18-Tat11-atto633/Cy3-plasmid DNA binary complexes at N:P ratio 16:1 applying the same treatments used for a typical transfection experiment. Scale bars: 50 µm. (f, g) Panels show the fluorescence signal distribution of CM18-Tat11-atto633 (f) and Cy3-plasmid (g) from the upper left quadrant of Figure 3e panels during the time-lapse. Scale bars: 10 µm. (h) Quantitative evaluation of CM18-Tat11-atto633 (red line) and Cy3-plasmid DNA (green line) signals during the time-lapse acquisition. (i) 24 hours CM18-Tat11-atto633 (f) and Cy3-plasmid (g) panels zoom merge. Scale bars: 10 µm.
Figure 4.
Acceptor photobleaching assay.
(a, b) acceptor photobleaching experiments on putatively non-transfected (a) or trasfected (b) cells. For both conditions, the first row of images shows CM18-Tat11-atto633 cell signal before and after a scanning bleaching of the whole cell with a 633nm laser at full power, while the second row shows the same for Cy3-plasmid DNA signal. Scale bar: 10 µm.
Figure 5.
In vitro peptide/DNA complex stability evaluation in endosomal-like conditions.
(a) EtBr exclusion assay performed as explained in caption 1b in PBS buffer at different pH: 7.4 (black column), 6.5 (dark grey), 5.5 (light grey), 4.5 (white). Y-axis break from 10% to 15% at 70% of the axis length. (b) EtBr exclusion assay 24 hours time-lapse performed at 37°C incubating CM18-Tat11/plasmid DNA binary complexes at pH=7.4 (black column) or pH=6.5 (red) with cathepsin B enzyme. (c) Graphical scheme of CM18-Tat11/plasmid DNA binary complexes disassembling evolution inside endosomal vesicle.