Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles

In
gene therapy, endosomal escape represents a major bottleneck
since nanoparticles often remain entrapped inside endosomes and are
trafficked toward the lysosomes for degradation. A detailed understanding
of the endosomal barrier would be beneficial for developing rational
strategies to improve transfection and endosomal escape. By visualizing
individual endosomal escape events in live cells, we obtain insight
into mechanistic factors that influence proton sponge-based endosomal
escape. In a comparative study, we found that HeLa cells treated with
JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape
frequency compared to ARPE-19 cells. We found that endosomal size
has a major impact on the escape capacity. The smaller HeLa endosomes
are more easily ruptured by the proton sponge effect than the larger
ARPE-19 endosomes, a finding supported by a mathematical model based
on the underlying physical principles. Still, it remains intriguing
that even in the small HeLa endosomes, <10% of the polyplex-containing
endosomes show endosomal escape. Further experiments revealed that
the membrane of polyplex-containing endosomes becomes leaky to small
compounds, preventing effective buildup of osmotic pressure, which
in turn prevents endosomal rupture. Analysis of H1299 and A549 cells
revealed that endosomal size determines endosomal escape efficiency
when cells have comparable membrane leakiness. However, at high levels
of membrane leakiness, buildup of osmotic pressure is no longer possible,
regardless of endosomal size. Based on our findings that both endosomal
size and membrane leakiness have a high impact on proton sponge-based
endosomal rupture, we provide important clues toward further improvement
of this escape strategy.