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

Abstract

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.

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

“…25 Other studies indicate that the actual mechanism could be much more complex than the classical proton sponge effect and may depend on fusion with the endosomal membrane and additional factors, such as the endosome size, membrane leakiness, late endosome formation, Rab7A localization on the surface of endosomes, and activation of mTORC1 for downstream signaling for protein synthesis. 26,27 Materials Used for Non-viral mRNA Delivery Ionizable Lipids. One well-studied class of non-viral mRNA delivery agents includes the cationic or ionizable lipids and lipid-like materials.…”

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“…[ 64 ] Recently, a report indicated that both endosomal size and membrane leakiness resulting from a difference in cell lines affected the proton sponge‐based endosomal rupture, providing important clues toward further improvement of this escape strategy. [ 65 ] The major concerns for the use of polycationic agents are toxicity‐induced acute inflammation, hemolysis, cell necrosis, apoptosis, [ 66 ] and low cytosol delivery, which result from the strong interaction between the polycationic agent and cell membranes. In addition, polycationic agents also have high nondegradability and suffer from low dissociation of the cargo from the complex in the cells due to robust interactions between the cargo and the polycation.…”

Gold Nanoparticle-Mediated Photoporation Enables Delivery of Macromolecules over a Wide Range of Molecular Weights in Human CD4+ T Cells

“…Viral vectors typically yield high transduction efficiencies, but their use is expensive, labor-intensive, and associated with significant safety issues [17,18]. While lipid-based and polymer-based nanoparticles can be safer alternatives, efficient transfection is often hindered by poor uptake, e.g., in blood and immune cells [14], limited endosomal escape [19,20], and delayed unpacking of the cargo molecules from the carriers in the cytosol [21]. In addition, these synthetic carriers can typically only be used with specific cargo molecules (e.g., nucleic acids) and cell types [22].…”