Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient

Abstract

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

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

“…Induction of vesicles with non-acidic pH could be crucial for the escape of CPP/cargo complexes from endosomal compartment [50]. On the other hand, CPPs may utilize two or more trafficking pathways depending on the experimental conditions [51]. Our studies refer to these observations as streptavidin transport by biotinylated TP and TP10 into CRC cell lines was independent of the endocytosis because brefeldin A did not inhibit this process.…”

Intracellular delivery of nanomaterials: How to catch endosomal escape in the act

“…Typically, though not always, these tracers are [55] and HPTS/DPX [55,56] have been used in ex cellulo assays to investigate the membrane integrity of artificial endosomes, where leakage and dilution of both compounds results in dequenching of the fluorescent signal. Fluorescein-labeled cell-penetrating peptides (CPPs) have been used in a similar manner with potassium iodide as quencher [57]. Otherwise, rather than using a quencher molecule, self-quenching of certain fluorophores can be achieved when used at a sufficiently high concentration, which is relieved upon leakage resulting in increased fluorescence.…”

“…As already highlighted, the leakage assay is frequently used to evaluate the effectiveness of certain compounds to induce pore formation, such as viral nanoparticles [61][62][63][64] and CPPs [53,55,[57][58][59]. Nonetheless, these assays were also used to verify the effectiveness of polymeric gene delivery complexes, composed of cationic polymers such as PEI [51,60] or poly(2-alkylacrylic acid) [47], and pDNA.…”

“…Upon illumination, BR will act as a proton-pumping V-type ATPase, hence acidifying the LUV's interior and mimicking the late endosomal environment in a controlled manner [57].…”

Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA

“…30,31 The other reason is that the high density of hydrophobic stearyl moieties increases the cellular uptake and fusion of complexes with the endosomal membrane by breaking it to facilitate endosomal escape. 32,33 Our cytotoxicity study demonstrated high biocompatibility of C-SHR/pGL3 and SHR/pGL3 complexes (Figure 7). Although the C-SHR/pGL3 complex has a higher molecular weight and charge density than the SHR/pGL3 complex, it did not induce additional cytotoxicity in cells.…”