The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

Abstract

Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

Figure 1

(A) Schematic evaluation of a single particle track from a set of confocal images acquired within 300 s, with a time lapse Δt = 1 s. Representative trajectories of complexes in not treated chinese hamster ovarian (CHO) cells: (B) Lipofectamine/DNA; (C) DOTAP/DOPC/DNA (DD/DNA). Representative trajectories of complexes in nocodazole (NCZ)–treated CHO cells: (D) Lipofectamine/DNA and (E) DD/DNA. Diffusion (red) and flow motion (blue) segments are shown. Relative populations of the acquired tracks for Lipofectamine and DD in not treated (F) and NCZ-treated (G) CHO cells. (H) Mean square displacement (MSD) analysis of two representative tracks. MSD calculation was used for the measurement of the dynamic parameters, i.e. diffusion coefficients and flow speed.

Figure 2. Colocalization of DNA-loaded complexes and endosomes.

(A) Representative confocal images of Cy3-labeled Lipofectamine/DNA (Lipofectamine) and DOTAP/DOPC/DNA (Control) complexes (‘red’ signal). In both images intracellular vesicles were labeled with the endosomial marker FM4–64 (‘green’ signal). In detail, after 1 h of incubation of cells with lipoplexes, the FM4–63 marker was added following manufacturer’s instructions. After 1 additional hour of incubation, cells were viewed at the confocal microscope. A single laser line (488 nm) was used to excite both fluorophores, whose emission was then recorded in two spectrally-separate channels (550–600 for Cy3 and 650–700 for FM4–64). (B) ImageJ software (NIH Image; http://rsbweb.nih.gov/ij/) was used to measure colocalization through Mander’s overlap coefficient (M1 = colocalized red/total red). Manders overlap coefficient M1 was calculated according to Costes et al. As evident, DNA was largely found inside endosomes. No significant differences between Lipofectamine and Control were found. Colocalization measurements were evaluated for not less than 50 complexes for each formulation.

Figure 3

Colocalization of Lipofectamine (A) and DOTAP/DOPC liposomes (i.e. control) (B) loaded with Cy3-labeled DNA (red) with Lysosome marker (green). (C) Percentage of DNA-loaded particles in the lysosomes. Transfection efficiency (TE) of Lipofectamine/DNA and control complexes in nocodazole- (D) and Ciliobrevin (D,E) treated CHO cells with respect to TE measured in untreated cells (TE_control).