Cellular internalization of eight LFcin derivatives.

<p>Cells were treated with 10 μM FITC-labeled LFcins for 1 h. The fraction of cells internalizing the LFcin was determined by flow cytometry. Statistical comparisons were performed by ANOVA. Significant differences from the uptake of HR9-FITC at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from seven independent experiments in each treatment group.</p

Noncovalent interactions between CPPs and plasmid DNA <i>in vitro</i>.

<p>(A) Gel retardation assay indicating the formation of CPP/DNA complexes. Various amounts of L6 or L5a were mixed with DNA at molecular N/P ratios of 0 (DNA only), 3, 6, 9, 12, 15, and 18. These complexes were analyzed by electrophoresis on a 0.5% agarose gel, followed by SYBR Safe staining. DNA images were captured using a ChemiDoc XRS+ Gel Imaging System (Bio-Rad). (B) The relative mobility of CPP/DNA complexes. The mobility of CPP/DNA complexes is indicated. Data are presented as mean ± SD from five independent experiments in each treatment group.</p

Influence of calcium chloride on CPP-mediated gene delivery.

<p>(A) Images of CPP-mediated delivery of the pEGFP-N1 plasmid DNA into cells in the absence or presence of calcium chloride. L6 or L5a was premixed with the pEGFP-N1 plasmid at an N/P ratio of 12. These CPP/DNA complexes, DNA alone, and CPP alone were incubated with cells for 1 h in the absence or presence of 113 mM CaCl₂. Cells without any treatments and cells treated with Lipofectamine 2000/DNA complexes served as negative and positive controls, respectively. BFP and GFP channels revealed the distribution of nuclei stained by Hoechst33342 and EGFP, respectively. Cell morphologies were observed in bright-field images. All images are obtained using a Motic AE31 fluorescent microscope. (B) Fluorescent intensity of CPP-mediated delivery of gene expression. Fluorescent intensity recorded in panel A was quantified using UN-SCAN-IT software. Statistical comparisons were performed by ANOVA. Data are presented as mean ± SD from nine independent experiments in each treatment group. Experimental groups were compared with the negative control, and each group without CaCl₂ treatment was compared with the group with CaCl₂ treatment. Significant differences at <i>P</i><0.05 (α) and <i>P</i><0.01 (**, αα) are indicated. (C) Transfection efficiency of CPP-mediated delivery of gene expression. Folds of transfection efficiency recorded in panel A were quantified using UN-SCAN-IT software. Data are presented as mean ± SD from nine independent experiments in each treatment group. Significant differences at <i>P</i><0.01 (**) are indicated.</p

Real-time PCR analysis of cellular delivery of reporter gene plasmid mediated by L6 and L5a.

<p>(A) Final RT-PCR products of <i>EGFP</i> and <i>18S rRNA</i> genes from the cells after uptake of the peptide/DNA complexes. Cells were not treated (negative control, NCtl) or were treated with DNA alone, CPP alone, Lipofectamine 2000/DNA (Lip/DNA) or CPP/DNA complexes for 24 h. Real-time PCR for the detection of <i>EGFP</i> gene expression was conducted using a Bio-Rad iQ5 Real-Time PCR Detection System. Human<i>18S rRNA</i> gene expression was analyzed as an internal control. Negative control (NTC) represents real-time PCR signal in the absence of a DNA template. (B) Real-time PCR analysis of <i>EGFP</i> gene expression. <i>EGFP</i> gene expression recorded in panel A was normalized to <i>18S rRNA</i> expression. Statistical comparisons were performed by ANOVA. Data are presented as mean ± SD from three independent experiments in each treatment group. Significant differences from cells without any treatments (negative control) at <i>P</i><0.01 (**) are indicated.</p

Fluorescent microscopy of cellular uptake of eight FITC-labeled LFcin derivatives into A549 cells.

<p>(A) Fluorescent images of cells treated with FITC-labeled LFcins. Cells were treated with 10 μM FITC-labeled LFcins for 1 h, and then stained with Hoechst 33342. Cells not exposed to LFcins served as internal controls. Cells were treated with HR9-FITC and bradykinin-FITC as positive and negative controls, respectively. GFP, BFP channels and bright fields revealed the distributions of FITC-labeled LFcins, nuclei and cell morphologies. All images are obtained using a Motic AE31 fluorescent microscope with a magnification of 400×. (B) Quantification of the uptake of FITC-labeled LFcins. The fluorescent intensity of cellular internalization was quantified using UN-SCAN-IT software. Each experiment group was compared with the internal control and statistical differences were calculated using the Student's <i>t</i>-test. Significant differences from HR9-FITC at <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from five independent experiments in each treatment group.</p

Zeta-potential and particle size analyses of CPPs, DNA, and CPP/DNA complexes.

<p>(A) Zeta-potentials of CPPs. HR9-FITC, bradykinin-FITC and eight FITC-labeled LFcin derivatives were dissolved in double deionized water and measured using a Zetasizer Nano ZS. Data are presented as mean ± SD from five independent experiments in each treatment group. (B) Zeta-potentials of DNA and CPP/DNA complexes. L6 and L5a were premixed with DNA at N/P ratios of 9 and 12. The DNA and CPP/DNA complexes were then dissolved in double deionized water and measured using a Zetasizer. Data are presented as mean ± SD from five independent experiments in each treatment group. (C) Particle sizes of DNA and CPP/DNA complexes. L6 and L5a were premixed with DNA at N/P ratios of 9 and 12. The DNA and CPP/DNA complexes were then dissolved in double deionized water and measured using a Zetasizer. Data are presented as mean ± SD from five independent experiments in each treatment group.</p

Confocal microscopy of intracellular delivery of IR/QD complexes into A549 cells.

<p>(A) Images of A549 cells treated with IR9/QD complexes prepared at various combination ratios. IR9 was mixed with QDs at molecular ratios of 120 and 240, and then incubated with cells for 24 h at 37°C. The cells were stained with Hoechst 33342 and then observed using a BD Pathway 435 System (BD Biosciences) at a magnification of 600×. GFP and BFP channels revealed the distribution of QDs and nuclei, respectively. Cell morphologies are shown in bright-field images. Overlaps between QDs and nuclei are cyan in merged GFP and BFP images. Scale bar is 25 µm. (B) Association between IR9 and QDs after cellular internalization. Cells were treated with IR9-FITC and QDr as controls. Five µM of IR9-FITC was mixed with 2.6 nM of QDr at 37°C for 2 h, and IR9-FITC/QDr complexes were added to cells for 24 h at 37°C. Cells were stained with Hoechst 33342 and observed using a Leica confocal microscope system at a magnification of 1,260×. GFP, RFP and BFP channels revealed the distribution of IR9-FITC, QDr and nuclei, respectively. Overlaps between peptides and QDr were yellow in merged GFP and RFP images. (C) Subcellular colocalization of IR9-delivered QDs. Cells were treated with IR9/QD complexes prepared at a molecular ratio of 120 for 24 h in the absence or presence of 25 µM chloroquine. Cells were stained with LysoTracker DND-99 and Hoechst 33342, and images were then observed using a BD Pathway System at a magnification of 600×. GFP, RFP and BFP channels displayed the distribution of QDs, lysosomes and nuclei, respectively. Overlaps between QDs and lysosomes were yellow/orange color in merged GFP and RFP images.</p

Noncovalent interactions between IR9 and QDs <i>in vitro</i>.

<p>(A) Gel retardation assay revealing stable interactions between IR9 and QDs. Different amounts of IR9 were mixed with QDs at molecular ratios of 0 (QD only), 15, 30, 60, 90 and 120. IR9/QD mixtures were subjected to electrophoresis on a 0.5% agarose gel. Fluorescence of QDs was visualized at 532 nm using a Typhoon Trio imager (GE Healthcare). (B) Relative shift of IR9/QD complexes formed at different IR9/QD ratios. Data are presented as mean ± SD from 6 independent experiments in each treatment group.</p

Noncovalent interactions between IR9 and plasmid DNAs <i>in vitro</i>.

<p>(A) Gel retardation assay of IR9/DNA complexes. Different amounts of IR9 were mixed with the pPEGFP-N1 plasmid at molecular ratios of 0 (DNA only), 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12 and 15, as indicated. After a 2 h incubation, IR9/DNA complexes were analyzed by electrophoresis on a 0.5% agarose gel and stained by SYBR® Green 1. (B) The relative shift percentage (<i>y</i>-axis) as a function of IR9/DNA ratio.</p

Zeta-potential measurements of QDs, IR9, DNAs and IR9/cargo complexes.

<p>(A) Zeta-potentials of QDs, IR9 and IR9/QD complexes. QDs, IR9 peptides or IR9/QD complexes prepared at a molecular ratio of 60 were liquefied with double deionized water and measured using a Zetasizer (Malvern Instruments). (B) Comparison of zeta-potentials of IR9/QD complexes prepared at molecular ratios of 60, 120 and 240, respectively. Samples were liquefied with double deionized water and measured using a zeta-potential analyzer. The value measured in IR9/QD complexes prepared at a molecular ratio of 60 served as the control. Zeta-potentials of IR9/QD complexes prepared at molecular ratios of 120 and 240 were compared to that of the control. Significant differences of <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from 3 independent experiments in each group. (C) Zeta-potentials of DNAs and IR9/DNA complexes. IR9 was mixed with the pEGFP-N1 plasmid at N/P ratios of 9 and 12, respectively. After forming complexes, DNA and IR9/DNA complexes were liquefied with double deionized water and measured using a zeta-potential analyzer. The value measured in IR9/DNA complexes at an N/P ratio of 9 served as the control. Significant differences of <i>P</i><0.01 (**) are indicated. (D) The correlation coefficient analysis between zeta-potential and protein transduction efficiency. Cells were treated with IR9/DNA complexes prepared at N/P ratios of 0 (DNA only), 3, 6, 9 and 12 and then analyzed using a Zetasizer. Transduction efficiency was analyzed by the UN-SCAN-IT software. A logarithmic curve was plotted with zeta-potential (<i>y</i>-axis) against transduction efficiency (<i>x</i>-axis) using SigmaPlot software.</p