Real-time gene delivery vector tracking in the endo-lysosomal pathway of live cells

Abstract

Using live-cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo-lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid-sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time post-transfection, gene vectors accumulate in late endosomes/lysosomes; however, real-time escape of vectors from membrane-bound vesicles is not observed.

Figure 1

Transport of PEI/DNA complexes in Lysotracker-stained vesicles of a live COS-7 cell. (A) PEI/DNA nanocomplexes (green) are found in acidic organelles (red), where yellow indicates co-localization. Late endosomes/lysosomes (LE/Lys) are stained with the live-cell dye Lysotracker. Line arrow and solid arrow indicate gene vectors co-localizing or not co-localizing with LE/Lys, respectively, with no obvious displacements from the starting positions. Empty arrowhead indicates a gene vector inside a LE/Lys that experiences a large displacement during the same elapsed time. Images are obtained from a 40 s-movie acquired at 2.5 frames/s. Bar is 5 µm. Also see Supplementary Material Movie 1. (B) Boxplots of normalized diffusivities of gene vectors and LE/Lys at a time scale of 10 s. Values were normalized to the geometric mean diffusivity value. The number of endosomes or PEI/DNA tracked are n = 73 (LE/Lys), n = 98 (PEI/DNA in LE/Lys), and n = 43 (PEI/DNA not in LE/Lys). There is no statistically significant difference between the groups as determined by ANOVA.

Figure 2

Per pixel co-localization of PEI/DNA complexes with either EE or LE/Lys at various times post-transfection in MSCs. To fluorescently label endosomes, cells were pre-transfected with GFP fusion genes of EEA1 (early endosome antigen 1) for EE or NPC1 (Niemann-Pick C1) for LE/Lys. Five cells from each condition were imaged.

Figure 3

Transfer of PEI/DNA nanocomplexes between EEs of a live Mesenchymal stem cell (MSC). Stills from a 60s-movie (obtained at 2.5 frames/s) show a PEI/DNA nanocomplex (indicated with an arrow) being transferred from one EE to another. EE are green due to the expression of EEA1-GFP, and PEI/DNA nanocomplexes are fluorescently labeled red with Alexa Fluor 546. Bar is 1 µm. Also see Supplementary Material Movie 2.

Figure 4

Transport of PEI/DNA nanocomplexes in late endosomes/lysosomes (LE/Lys) of a live MSC. (A) Many PEI/DNA complexes (red) co-localize with NPC-1 (green) at 2 h post-transfection. Boxes indicate regions shown in greater detail in panels B or C. Bar is 10 µm. Also see Supplementary Material Movie 3. (B) PEI/DNA complex moving within a tubular LE. Bar is 1 µm. (C) PEI/DNA (indicated by an arrow) moving actively through a tubule that is lined with NPC1. Bar is 1 µm. Movies captured at 2.5 frames/s.

Figure 5

Transfer of PEI/DNA between LE/Lys of a live MSC. Stills from a 60s-movie (obtained at 2.5 frames/s) showing a PEI/DNA nanocomplex (indicated by an arrow) being transferred from one LE/Lys to another. LE/Lys are green due to the expression of NPC1-GFP, and PEI/DNA nanocomplexes are fluorescently labeled red with Alexa Fluor 546. Bars are 1 µm. Also see Supplementary Material Movie 4.

Figure 6

Boxplots of normalized diffusivities of PEI/DNA complexes transporting within EE or LE/Lys at a time scale of 10 s. Values were normalized to the geometric mean diffusivity value. The number of PEI/DNA tracked are n = 81 (PEI/DNA in EE) and n = 61 (PEI/DNA in LE/Lys). There is no statistically significant difference between the groups as determined by student t-test.