Scavenger receptor‐mediated uptake of cell‐penetrating peptide nanocomplexes with oligonucleotides

Abstract

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.

Peptides for nucleic acid delivery

“…It was found that the zeta potential of PepFect nanocomplexes is positive only at high peptide:nucleic acid ratios and when measured in water. However, when measured in isotonic media containing ions or serum-free media, the net charge of these complexes is predominantly negative [108]. Zeta potential is a function of the ion concentration and the pH of the medium, and it changes due to the adherence of a layer of counter ions on the surface of the nanostructure which is called the Stern layer [109].…”

“…This led to the assumption that a receptor might be involved in the uptake as the direct interaction of the newly formed anionic surface with the negatively charged plasma membrane or HS proteoglycans would lead to electrostatic repulsion. Using competitive inhibitors, siRNA knockdown and colocalization experiments, it was found that class A scavenger receptors, SCARA3 and SCARA5, are involved in the uptake of these nanocomplexes [108]. This class of innate immune receptors is known to bind negatively charged particulate ligands [110].…”

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

“…SASH1 gene has been chosen as a target for siRNA delivery by using TP or TP10, since our preliminary data showed a stable expression of this gene in HT29 and HCT116 cells. We used a procedure described by Ezzat et al [31]. Cells were seeded into 24-well plates at a density of 50 000 cells/well in 1 mL of medium.…”

Peptide-based vectors: recent developments

“…It is important to study the uptake mechanism and the biological effects of the complexes instead of the peptide alone. In 2012, the involvement of scavenger receptor class A (SCARA) in the uptake of noncovalent CPP:ON complexes was shown for the first time (42). It was previously shown that SCARA receptors bind and mediate cellular uptake of a negatively charged molecules (43), and the involvement of SCARA was shown for the CPP PepFect14 and splice-correcting oligonucleotides (SCOs), which have negative ζ potential and NF51/pDNA (44).…”