PepFect6 Mediated SiRNA Delivery into Organotypic Cultures

Abstract

Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and nonviral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response.

Improved Cellular Delivery of Antisense Oligonucleotide for miRNA-21 Imaging In Vivo Using Cell-Penetrating Peptide-Based Nanoprobes

“…Cell-penetrating peptides (CPPs) are a class of peptides that have functioned as potential vectors for delivering nucleic acids and other bioactive molecules in vitro and in vivo. , Recently, a novel CPP PepFect6 (PF6) has been constructed by adding stearyl and trifluoromethylquinoline moieties into the widely used CPP transporter 10 (TP10). − Studies confirm that stearylation reduces toxicity and improves transfection efficiency, while the lysosomotropic agent trifluoromethylquinoline enhances the endosomal release of the oligonucleotide . The nanoparticle formation of oligonucleotides and PF6 has suggested promising perspectives in therapeutic applications. ,, However, the value of the AMO/PF6 nanoparticle in antisense imaging is still unknown.…”

“…CPPs are short cationic and amphipathic peptides that can gain access to cells and, importantly, promote the cellular uptake of various bioactive cargos . PepFects (PFs), a group of CPPs usually <30 amino acids in length, have been shown to achieve high-efficiency nucleic acid delivery with minimal cytotoxicity and immunogenicity. , Recently, PF6 has been proven to be capable of delivering small nucleic acid molecules such as siRNAs and oligonucleotides into cells. ,, The high delivery efficiency of PF6 benefits from its proper chemical modifications. ,,,, First, the introduction of a stearic acid moiety to the N-terminus of TP10 increases the uptake of AMO and improves the stability of the peptide in serum. The stearylation of the CPPs adds hydrophobicity to peptides, giving complexes a more lipophilic nature, further facilitating the penetration through the cell membrane and enabling stronger hybridization with AMOs.…”

“…15,24 Recently, PF6 has been proven to be capable of delivering small nucleic acid molecules such as siRNAs and oligonucleotides into cells. 16,18,19 The high delivery efficiency of PF6 benefits from its proper chemical modifications. 13,15,16,20,25 First, the introduction of a stearic acid moiety to the N-terminus of TP10 increases the uptake of AMO and improves the stability of the peptide in serum.…”

“…17 The nanoparticle formation of oligonucleotides and PF6 has suggested promising perspectives in therapeutic applications. 16,18,19 However, the value of the AMO/PF6 nanoparticle in antisense imaging is still unknown.…”

Innovative pharmaceutical approaches for the management of inner ear disorders

“…The diffused interest for the therapeutic applications of nanotechnology has also driven the research to novel drug delivery systems intended for both intra-tympanic and intra-cochlear administration. Considering the ability of nanoparticles to control and target the drug release, polymeric nanoparticles [40,[98][99][100][101][102][103][104][105][106], lipid nanocapsules [107,108], cubosomes [109], polyplexes [110,111], metallic core nanoparticles [38,[112][113][114][115], liposomes [43,[116][117][118] and lipoplexes [119] were investigated in vitro and in vivo as drug-delivery systems or imaging agents.…”