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.
|Titolo:||The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery|
|Data di pubblicazione:||2016|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1038/srep25879|
|Parole Chiave:||Animals; Biological Transport; CHO Cells; Cell Nucleus; Cricetulus; Cytoplasm; DNA; Genetic Therapy; Genetic Vectors; Humans; Lipids; Single Molecule Imaging; Transfection; Multidisciplinary|
|Appare nelle tipologie:||1.1 Articolo in rivista|