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.

Muography is an imaging technique based on the measurement of absorption profiles for muons as they pass through rocks and earth. Muons are produced in the interactions of high-energy cosmic rays in the Earth’s atmosphere. The technique is conceptually similar to usual X-ray radiography, but with extended capabilities of investigating over much larger thicknesses of matter thanks to the penetrating power of high-energy muons. Over the centuries a complex system of cavities has been excavated in the yellow tuff of Mt. Echia, the site of the earliest settlement of the city of Naples in the 8th century BC. A new generation muon detector designed by us, was installed under a total rock overburden of about 40 metres. A 26 days pilot run provided about 14 millions of muon events. A comparison of the measured and expected muon fluxes improved the knowledge of the average rock density. The observation of known cavities proved the validity of the muographic technique. Hints on the existence of a so far unknown cavity was obtained. The success of the investigation reported here demonstrates the substantial progress of muography in underground imaging and is likely to open new avenues for its widespread utilisation.

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