Mechanism of cellular internalization of PR9/QD complexes.

<p>(A) Cellular uptake of PR9/QD complexes without or with inhibitors. Cells were treated with PBS as a negative control, QDs alone or PR9/QD complexes in the absence or presence of endocytic inhibitors. Low temperature (4°C), 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)-amiloride (EIPA), cytochalasin D (CytD), filipin and nocodazole inhibit different endocytic pathways. Flow cytometry was used to quantitate transduction efficiency. (B) Effect of the lysosomotropic agent chloroquine on cellular uptake of CPP/QD complexes. Significant differences at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from seven (A) and eleven (B) independent experiments.</p

Intracellular trafficking of PR9/QD complexes.

<p>(A) Trafficking of PR9/QD complexes toward early endosomes. Cells were treated with PR9/QD complexes for 30 min to 5 h and stained with anti-human early endosome antigen 1 protein (EEA1) antibody. Overlaps of green fluorescent QDs and red fluorescent early endosomes are yellow in merged GFP and RFP images. (B) Trafficking of PR9/QD complexes toward lysosomes. Cells were treated with PR9/QD complexes for 30 min to 5 h and stained with LysoTracker DND-99 and Hoechst 33342. (C) Trafficking of PR9/QD complexes toward the nucleus. A549 cells were treated with PR9/QD complexes for 30 min to 5 h and stained with Hoechst 33342. (D) Time course of colocalization of PR9/QD complexes with lysosomes. (E) Time course of colocalization of PR9/QD complexes with nuclei. Significant differences at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from seven independent experiments. Cell morphology is shown as bright-field images. All fluorescent (A) and confocal images (B and C) are shown at a magnification of 600×.</p

Cytotoxicity of materials used for cargo delivery by PR9 as determined using the MTT assay.

<p>Cells were treated with QD, PR9, chloroquine, PR9/QD, QD/chloroquine, PR9/QD/chloroquine, QInP alone and PR9/QInP complexes for 24 h. Significant differences at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from three independent experiments.</p

Zeta-potential and particle size of PR9 and PR9/QD complexes and the secondary structure of PR9.

<p>(A) Zeta-potentials of PR9 and PR9/QD complexes. PR9 or PR9/QD complexes prepared at a molecular ratio of 60 were dissolved in doubly deionized water at pH 7 or 5. Each solution was equilibrated at 25°C for 120 sec in a zeta cell and then analyzed using a Zetasizer Nano ZS. (B) Particle size of QD or PR9/QD complexes. PR9/QD complexes were dissolved in doubly deionized water with pH 7 or 5 and then analyzed using a Zetasizer. Significant differences between PR9/QD complexes and QDs at <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from seven independent experiments. (C) Secondary structure of PR9. All CD spectra were recorded in millidegree (mdeg).</p

Transmission electron microscopy (TEM) images of PR9/QD complexes.

<p>(A) Images of PR9/QD complexes and PR9/QD-transduced cells. PR9 peptide was mixed with QDs at a molecular ratio of 60 (left). Cells were treated with PR9/QD complexes; arrows indicate the location of PR9/QD complexes (middle and right). (B–F) Images of PR9/QD-transduced cells. CCP = clathrin-coated pit, EE = early endosome, ER = endoplasmic reticulum, Ly = lysosome, Ma = macropinosome, MP = membrane protrusion, Mt = mitochondrium, N = nucleus, PM = plasma membrane, V = vesicle.</p

Different cargoes delivered by PR9 follow similar trafficking route in cells.

<p>(A) Intracellular trafficking of PR9/QInP complexes. A549 cells were treated with PR9/QInP complexes for 30 min to 5 h and stained with LysoTracker DND-99 and Hoechst 33342. All confocal images are shown at a magnification of 600×. (B) Functional assay of the PR9-delivered plasmid DNA in cells. Cells were treated with pEGFP-N1 plasmid DNA alone or PR9/DNA complexes for 24 h followed by the stain with Hoechst 33342. Green fluorescence revealed cells expressing EGFP (only one of many EGFP-expressing cells is shown here). Blue fluorescence indicates nuclei. Images are shown at a magnification of 200×. (C) Time course of gene expression for PR9/DNA complexes. Significant differences at <i>P</i><0.05 (*) are indicated. Data are presented as mean ± SD from three independent experiments.</p

Time course analysis of CPP-mediated cellular internalization of QDs.

<p>CPPs, including SR9, HR9 and PR9, were premixed with QDs and then incubated with A549 cells for 0, 0.5, 1, 2, 3, 4, 5 and 6 h. Cellular internalization efficiency was analyzed by flow cytometry. Cells shown green fluorescence were counted as positive signals. Data are presented as mean ± SD from seven independent experiments.</p

Intracellular colocalization of CPP/QD complexes with organelles.

<p>A549 cells were treated with QDs alone, SR9/QD, HR9/QD or PR9/QD complexes for 30 min or 2 h and then stained with Texas Red-X phalloidin for actin filaments (A), LysoTracker DND-99 for lysosomes (B), MitoTracker Deep Red FM for mitochondria (C) or ER-Tracker Red for endoplasmic reticula (ER) (D). Nuclei were stained with Hoechst 33342. Overlap of QDs and organelle trackers are yellow in merged GFP and RFP images. Overlaps of QDs and nuclei are cyan in merged GFP and BFP images. All fluorescent images (A–D) are shown at a magnification of 600×.</p