Despite recent advances in the field, confusions surrounding CPP internalization mechanisms and intracellular trafficking are hindering the development of new and more efficient vectors. Nevertheless, the recent increase in the number of publications containing in vivo CPP utilization looks promising that the number of clinical trials would also increase in the near future.
Cell-penetrating peptides have been extensively used since their discovery for delivering cargoes unable to cross the cell membrane. Among other transported cargoes, they have shown very efficient delivery for oligonucleotides making cell-penetrating peptides a powerful tool for gene therapy. Numerous cell-penetrating peptides have now been discovered offering a wide library of structures and mechanisms of actions. Nevertheless, if it is known that different pathways are available for particles to be taken up, most mechanisms by which these particles enter cells are still to be characterized more precisely. Indeed it is admitted that cell-penetrating peptides are taken up either by direct translocation or by endocytosis but classes of cell-penetrating peptides are usually not related to specific entrance mechanisms. Actually, for most particles, different pathways can be detected during their uptake which makes the literature sometimes contradictory. Recent studies have nevertheless shown convergent uptake patterns for individual structures. Acetylated cell-penetrating peptides complexed with oligonucleotides have been shown to interact to scavenger receptor class A to induce caveolae-mediated endocytosis whereas antimicrobial peptides create pores in the cell membrane for direct translocation. Arginine-rich peptides have presented concentration-dependent mechanisms, being taken up either by membrane destabilization or clathrin-mediated endocytosis. Relating the structure of cell-penetrating peptides or their particles to distinct mechanisms would allow this delivery platform to become even more specific by using rational design to fit to the desired uptake pathway.
Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.
Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our...
Cell-penetrating peptides (CPPs) are short peptides that are able to efficiently penetrate cellular lipid bilayers. Although CPPs have been used as carriers in conjugation with certain cargos to target specific genes and pathways, how rationally designed CPPs per se affect global gene expression has not been investigated. Therefore, following time course treatments with 4 CPPs-penetratin, PepFect14, mtCPP1 and TP10, HeLa cells were transcriptionally profiled by RNA sequencing. Results from these analyses showed a time-dependent response to different CPPs, with specific sets of genes related to ribosome biogenesis, microtubule dynamics and long-noncoding RNAs being differentially expressed compared to untreated controls. By using an image-based high content phenotypic profiling platform we confirmed that differential gene expression in CPP-treated HeLa cells strongly correlates with changes in cellular phenotypes such as increased nucleolar size and dispersed microtubules, compatible with altered ribosome biogenesis and cell growth. Altogether these results suggest that cells respond to different cell penetrating peptides by alteration of specific sets of genes, which are possibly part of the common response to such stimulus.
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