Macropinocytosis-like endocytic mechanism contributes to the overall internalization of polyethyleneimine polyplexes used in nucleic acid delivery
This study reveals that Linear Polyethyleneimine (LPEI) polyplexes enter HeLa cells primarily through a dynamin-dependent, non-canonical macropinocytosis-like mechanism characterized by membrane ruffling, which operates alongside overlapping endocytic pathways to facilitate efficient nucleic acid delivery.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer
To deliver life-saving genetic instructions into a human cell, scientists often use tiny molecular packages called polyplexes. These packages are formed when a synthetic, positively charged material called polyethyleneimine wraps around a strand of genetic code, creating a compact unit small enough to enter a cell. However, getting inside is only the first hurdle; the cell must actively swallow the package for the treatment to work. This swallowing process, known as endocytosis, is not a single, simple door but a complex network of different entry routes. Some routes use specific coated pits to grab items, while others rely on the cell membrane stretching out like a hand to scoop up fluids and particles. Understanding exactly which route a drug carrier takes is critical, because the path it chooses determines whether it successfully delivers its cargo or gets destroyed along the way.
In a recent study, researchers investigated how these polyplexes enter human cells, specifically looking at a type of cell line often used in laboratories. They began by testing whether the cells needed energy to swallow the packages. When they cooled the cells down to a temperature where cellular activity slows to a near halt, the polyplexes barely entered at all. Similarly, when they chemically blocked the cells' ability to produce energy, the uptake dropped dramatically. This confirmed that the process is an active, energy-driven event, not a passive drifting of particles into the cell. The team then tried to block specific, known entry routes one by one. They used chemicals to stop the coated-pit mechanism, the cave-like invaginations, and the fluid-scooping mechanism known as macropinocytosis. Surprisingly, blocking any single one of these pathways did not stop the polyplexes from entering. The cells seemed to have a backup plan, shifting their internal machinery to use a different route whenever one was blocked, ensuring the packages still got inside.
To see exactly what was happening on the surface of the cell, the researchers turned to powerful electron microscopes, which provide a view far sharper than standard light microscopes. They observed that the polyplexes did not settle into small, neat pits as one might expect. Instead, they gathered in large clusters that triggered the cell membrane to ripple and fold over them. These ripples looked very much like the membrane movements seen during macropinocytosis, a process where the cell engulfs large volumes of fluid. However, when the researchers looked closer at the internal structures after the packages were swallowed, they found something unexpected. The polyplexes were not trapped in the huge, irregular bubbles typical of standard macropinocytosis. Instead, they were wrapped in smaller, more uniform vesicles. This suggested the cells were using a specialized, non-standard version of the ruffling mechanism, one that is more precise and targeted than the usual fluid-scooping method.
The study also revealed that this unique entry route relies on a specific molecular machine called dynamin, which acts like a pair of molecular scissors to pinch off the new vesicles from the cell membrane. When the researchers used a drug to disable dynamin, the uptake of the polyplexes plummeted by nearly eighty percent. This finding was crucial because it showed that while the cells could bypass many other blocked pathways, they could not bypass the need for this specific pinching action. The researchers concluded that the polyplexes enter through a distinct, ruffle-mediated mechanism that looks like macropinocytosis but operates with different internal rules. It is a flexible, energy-dependent process that allows the cell to swallow these genetic carriers efficiently, provided the molecular scissors are working. This discovery helps scientists understand the complex logistics of getting genetic medicine into the body, suggesting that future designs might need to target these specific ruffling mechanisms to improve delivery.
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