Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus

Abstract

It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)-tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.

Figure 1

Effect of nocodazole on transfection activity of R8-Lip and adenovirus. (a) R8-Lip and (b) adenovirus containing genes encoding luciferase were transfected to HeLa cells for 6 hours in the absence or presence of nocodazole. Each bar represents mean ± SD of N = 3 experiments. *Significant differences determined by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison post hoc test. Differences against nocodazole-treated group were considered significant at P < 0.05. RLU, relative light unit.

Figure 2

Effect of nocodazole on cellular uptake and transcription activity. (a) R8-Lip (black bars) and adenovirus (white bars) were transfected to HeLa cells in the presence or absence of 10 µmol/l nocodazole for 3 hours. DNA was purified from cell lysate and then subject to the real-time PCR to measure the cellular uptake by means of the number of gene copies. (b) pDNA encoding luciferase was injected to the nucleus with injection marker (RhoDex) in the absence or presence of nocodazole. Transfection activity was evaluated after 6 hours. The activity was normalized by the number of nuclear RhoDex-positive cells. Each bar represents mean ± SD of N = 3 experiments. Significant difference was not observed between nocodazole-treated group and nontreated group in R8-Lip and adenovirus. RLU, relative light unit.

Figure 3

Imaging of R8-Lip and adenovirus for the analysis of intracellular trafficking. (a,b) R8-Lip encapsulating rhodamine-labeled pDNA (a) and Texas Red–labeled adenovirus (b) were transfected to GFP/Tub-HeLa cells for 1, 2, and 3 hours (represented in left, middle, and right panels, respectively). (c,d) A series of time-lapse images for R8-Lip (c) and adenovirus (d) in the process of directional transport is shown. Images shown for 10-second and 4-second frame intervals are represented, respectively. The original videos are represented in Supplementary Video S1. In the far right column, overlay images are represented to show the trajectories. GFP, green fluorescent protein.

Figure 4

Quantitative particle tracking analysis for intracellular dynamics of R8-Lip and adenovirus. (a,b) Typical trajectories for R8-Lip (a) and adenovirus (b) were represented. Frames subject to the directional transport are shown as red points and lines. These trajectories are obtained by 10-second and 2-second frame intervals, respectively. Rhodamine-pDNA and Texas Red–labeled adenovirus are represented as red. GFP/Tub is represented as green. (c,d) n = 28 and n = 34 MSD-Δt fitting curves for directional transport processes of R8-Lip (c) and adenovirus (d) were displayed, respectively. (e) The average velocity (v) obtained by the curve fitting of MSD-Δt curves for quasi-straight trajectories of R8-Lip (n = 28) and adenovirus (n = 34) was plotted. Black bars represent the mean values. Asterisks represent a significant difference, as determined by the Mann–Whitney test (**P < 0.01). GFP, green fluorescent protein; MSD, mean-square displacements.

Figure 5

Quantitative particle tracking analysis for diffusive motion of R8-Lip and adenovirus. (a,b) Typical trajectories for R8-Lip (a) and adenovirus (b) in microtubule-disruptive condition. R8-Lip and adenovirus are transfected to the GFP/Tub-HeLa cells in the presence of nocodazole. The time-lapse images were acquired by 10-second and 2-second frame intervals, respectively. The videos are represented in Supplementary Video S2. (c) The average diffusion constant (D) obtained by the linear fitting of MSD-Δt curves for diffusive motion of R8-Lip (n = 19) and adenovirus (n = 16) was plotted in right panel. The values determined by trajectories in diffusive motion in nocodazole-untreated cells (as typically represented in Figure 4a,b; black dots and lines) were plotted in the left panel. Black bars represent the mean values. Asterisks represent a significant difference, as determined by the Mann–Whitney test (**P < 0.01). GFP, green fluorescent protein; MSD, mean-square displacements.

Figure 6

Time-lapse imaging of QD-labeled pDNA encapsulated in rhodamine-labeled lipid envelope. Dually labeled R8-Lip, in which pDNA was labeled by QD705 and lipid envelope was labeled by rhodamine, was transfected to the GFP/Tub-HeLa cells. The time-lapse images were acquired by 5-second frame intervals. QD705, GFP/Tub, and rhodamine were represented as blue (B), green (G), and red (R) colors, respectively (top). To clearly show the colocalization of QD705 and rhodamine, dual color images for B+G (middle) and G+R (bottom) were also represented. GFP, green fluorescent protein.

Figure 7

Dual imaging of vesicular transport and carriers. (a,b) Typical images for the directional transport of rhodamine-pDNA-encapsulation R8-Lip (a) and Texas Red–labeled adenovirus (b) transfected in the presence of AFD488. Rhodamine-pDNA and Texas Red–labeled adenovirus are represented as red. Endosomes stained with AFD488 are represented as green. In the rightmost column, overlay images were represented to show the trajectories. The time-lapse images were shown by 4-second and 2-second frame intervals, respectively. (c) The average velocity (v) obtained by the curve fitting of MSD-Δt curves for quasi-straight trajectories of endosomes with (n = 12) and without (n = 14) carrying R8-Lip was plotted. Black bars represent the mean values. Asterisks represent a significant difference, as determined by the Mann–Whitney test (**P < 0.01). GFP, green fluorescent protein; MSD, mean-square displacements.