Polyplexes Are Endocytosed by and Trafficked within Filopodia

Abstract

The improvement of nonviral gene therapies relies to a large extent on understanding many fundamental physical and biological properties of these systems. This includes interactions of synthetic delivery systems with the cell and mechanisms of trafficking delivery vehicles, which remain poorly understood on both the extra- and intracellular levels. In this study, the mechanisms of cellular internalization and trafficking of polymer-based nanoparticle complexes consisting of polycations and nucleic acids, termed polyplexes, have been observed in detail at the cellular level. For the first time evidence has been obtained that filopodia, actin projections that radiate out from the surface of cells, serve as a route for the direct endocytosis of polyplexes. Confocal microscopy images demonstrated that filopodia on HeLa cells detect external polyplexes and extend into the extracellular milieu to internalize these particles. Polyplexes are observed to be internalized into membrane-bound vesicles (i.e., clathrin-coated pits and caveolae) directly within filopodial projections and are subsequently transported along actin to the main cell body for potential delivery of the nucleic acids to the nucleus. The kinetics and speed of polyplex trafficking have also been measured. The polyplex-loaded vesicles were also discovered to traffic between two cells within filopodial bridges. These findings provide novel insight into the early events of cellular contact with polyplexes through filopodial-based interactions in addition to endocytic vesicle traffickingan important fundamental discovery to enable advancement of nonviral gene editing, nucleic acid therapies, and biomedical materials.

Physicochemical properties and bio‐interfacial interactions of surface modified PDLLA‐PAMAM linear dendritic block copolymers

“…The cationic nature of the nanoaggregates creates favorable electrostatic interactions with the negatively charged cellular membrane, inducing uptake. The route of uptake observed here is similar to that observed by Reineke et al, 54 where polyplexes were uptaken into cells via the filopodia, then trafficked into the lamellipodia. The confocal images (Figure 5(A)) indicate that the cationic nanoparticles are being up‐taken via endocytosis by the filopodia, the actin projections that extend from the surface of the HEK cells.…”

Heteroacene-Based Amphiphile as a Molecular Scaffold for Bioimaging Probes

“…The cationic surface charge of TRPZ-bisMPA NPs leads to apparent cellular uptake via electrostatic interactions with anionic cell membranes. Similar results have been observed with other dendrimer-based NPs modified with anime groups (Morris et al, 2017;Ray et al, 2018;Ingle et al, 2020). Likely owing to the polar groups on the MPA dendron, TRPZ fluorescence is observed throughout the interior of the lysosome (Figure 4D).…”

Polymeric Delivery of Therapeutic Nucleic Acids

“…Outside PEI, Reineke and coworkers tracked the filopodia-driven transport of polyplexes formulated from the cationic glycopolymer Glycofect and concluded that these complexes were trafficked over long distances (13 μm) along filopodial projections at a velocity from 0.003 to 0.07 μm•s −1 . 255 There is debate about the timing of DNA release by polycations, and it is unclear if polyplexes outside of endosomes are efficiently trafficked. There is evidence, however, that some naked plasmids (i.e., plasmids uncomplexed from the polycation) can utilize intracellular machinery in the cytoplasm to complete the race to the nucleus and allow for nuclear uptake.…”