The role of endocytosis in the uptake and intracellular trafficking of PepFect14–nucleic acid nanocomplexes via class A scavenger receptors

Abstract

Cell penetrating peptides are efficient tools to deliver various bioactive cargos into cells, but their exact functioning mechanism is still debated. Recently, we showed that a delivery peptide PepFect14 condenses oligonucleotides (ON) into negatively charged nanocomplexes that are taken up by cells via class A scavenger receptors (SR-As). Here we unraveled the uptake mechanism and intracellular trafficking of PF14-ON nanocomplexes in HeLa cells. Macropinocytosis and caveolae-mediated endocytosis are responsible for the intracellular functionality of nucleic acids packed into nanocomplexes. However, only a negligible fraction of the complexes were trafficked to endoplasmic reticulum or Golgi apparatus - the common destinations of caveolar endocytosis. Neither were the PF14-SCO nanocomplexes routed to endo-lysosomal pathway, and they stayed in vesicles with slightly acidic pH, which were not marked with LysoSensor. "Naked" ON, in contrary, was rapidly targeted to acidic vesicles and lysosomes. The transmission electron microscopy analysis of interactions between SR-As and PF14-ON nanocomplexes on ultrastructural level revealed that nanocomplexes localized on the plasma membrane in close proximity to SR-As and their colocalization is retained in cells, suggesting that PF14-ON complexes associate with targeted receptors.

Cell‐penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids

“…Recently, we showed that PF14-SCO complexes can harness type A SRs to enter the cells (7), which appeared to be a more general cellular uptake mechanism for CPPs and their complexes with NAs (2,(10)(11)(12)(13). Surprisingly, we recently discovered that the particular receptors that are involved in CPP-mediated delivery (SR-A3 and SR-A5) do not localize in the plasma membrane of cells but are recruited there after the exposure to PF14-SCO nanocomplexes (5). In this work we aimed to unravel the mechanisms behind the relocation of SR-As from membranous structures from the cells' cytoplasm to the plasma membrane.…”

“…PI3Ks are a family of signaling proteins involved in the regulation of multiple cellular processes, among which the regulation of intracellular vesicle trafficking is relevant for our study. PI3Ks also contribute to the macropinosome formation and entry via micropinocytosis, which is also the main uptake mechanism for PF14-SCO nanocomplexes (5). Moreover, PI3K activity and its signaling can be controlled by calcium ions (48,49), which are required for SR-Amediated uptake of PF14-SCO nanocomplexes.…”

“…When studying NA delivery with PepFects, we discovered that SR-A3s and SR-A5s do not localize on the plasma membrane of untreated HeLa cells but are quickly recruited there after exposure to PF14-SCO nanocomplexes (5). However, their concentration in the cell cytoplasm is rather high, as revealed by electron and confocal microscopy (Supplemental Fig.…”

“…These results suggest that SR-dependent internalization is a rather general mechanism for the cellular uptake of CPP-NA nanoparticles and for polycationic agents used for transfection of cells. Our recent studies on the involvement of SR-A3s and SR-A5s in the uptake of PF14-SCO complexes revealed that these receptors are not constantly present on the plasma membrane of HeLa pLuc cells but are targeted there after exposure to the nanocomplexes, whereas the number of these receptors inside the HeLa pLuc cells was found to be rather high (5).…”

“…Despite having been studied for years, the exact mechanism by which CPPs and their complexes with NAs enter cells and exert their activity is only starting to unravel. The plasma membrane of cells regulates the trafficking of biomolecules between the extracellular and the intracellular environment, and association of a CPP or its construct with cargo to the cell surface induces its internalization via all major endocytic pathways described so far (1)(2)(3)(4)(5). However, some CPPs complexed with cargo molecules are suggested to enter the cells in an energy-independent manner via direct translocation across the plasma membrane of the cells (6).…”