Arginine-rich, cell penetrating peptide–anti-microRNA complexes decrease glioblastoma migration potential

Abstract

MicroRNAs (miRNAs) are a class of gene regulators originating from non-coding endogenous RNAs. Altered expression, both up - and down-regulation, of miRNAs plays important roles in many human diseases. Correcting miRNA dysregulation by either inhibiting or restoring miRNA function may provide therapeutic benefit. However, efficient, nontoxic miRNA delivery systems are in need. Cell penetrating peptides (CPPs) have been widely exploited for protein, DNA, and RNA delivery. Few have examined CPP transfection efficiency with single stranded anti-miRNA. The R8 peptide condensed both siRNA and anti-miRNA. Greater than 50% of cells had anti-miRNA/R8 complexes associated and in these cells 68% of anti-miRNA escapes the endosome/lysosome. Single -stranded antisense miR-21 inhibitor (anti-miR-21) administered using the R8 peptide elicited efficient downstream gene upregulation. Glioblastoma cell migration was inhibited by 25% compared to the negative control group. To our knowledge, this is the first demonstration of miRNA modulation with anti-miR-21/R8, complexes which has laid the groundwork for further exploring octaarginine as intracellular anti-miRNAs carrier.

Attachment of Cell-Binding Ligands to Arginine-Rich Cell-Penetrating Peptides Enables Cytosolic Translocation of Complexed siRNA

“…GFP siRNA complexed to 9DR or 9LR did not silence GFP expression in Neuro2a cells stably expressing the protein (Figures 1G and 1H). Therefore, 9R peptides poorly translocated siRNA, unless used at a high molar excess [>50, (Cantini, et al, 2013; Wang, et al, 2007; Zhang, et al, 2014)].…”

“…Despite their typical ability to effectively translocate biological macromolecules, R-CPPs are poor vehicles for cytoplasmic delivery of siRNA. For measurable mRNA knockdown, a huge excess of CPP molecules and high siRNA concentrations (above the therapeutic range) and/or association with reagents that disrupt endosomes is generally necessary (Akita, et al, 2010; Cantini, et al, 2013; El-Sayed, et al, 2009; Endoh and Ohtsuki, 2009; Erazo-Oliveras, et al, 2012; Lee, et al, 2008; Margus, et al, 2012; van Asbeck, et al, 2013; Zhang, et al, 2014). Imaging studies reveal the vast majority of CPP-siRNA complexes trapped for extended time periods in intracellular vesicles with little or no cytoplasmic localization (Al-Taei, et al, 2006; El-Sayed, et al, 2009; Erazo-Oliveras, et al, 2012; Fretz, et al, 2007; Fuchs and Raines, 2004; Maiolo, et al, 2005; Verdurmen, et al, 2011).…”

Charged group surface accessibility determines micelleplexes formation and cellular interaction

“…With the similar charge and size, the oligoarginine available on the micelle surface was expected to influence the ability of micelles to associate with or enter cells, but the total arginine and surface charge was not directly related to cellular interaction. 24, 40, 41 Only the available arginine content on the micelle surface correlated with the ability of the micelles to interact with cells. This further supports the idea that the oligoarginine on the mPEG-PLA-R 15 micelles was more available for interactions.…”

RNA interference for glioblastoma therapy: Innovation ladder from the bench to clinical trials

“…CPPs are small peptides containing 6–30 amino acids that are capable of membrane translocation and internalization by adsorptive mediated endocytosis [137]. RNA oligonucleotides can form complexes with peptides such as octoarginine (R8) which can aid in their cellular internalization and endosomal escape in comparison to unmodified RNAi molecules [137]. Consequently, CPPs could increase the RNAi therapeutic effect.…”

“…Consequently, CPPs could increase the RNAi therapeutic effect. For example, Y. Zhang et al showed that antimir-21/R8 treatment of U251 human GBM cells led to the downregulation of the well-known miR-21-regulated genes, PDCD4 and SERPINB5; as well as inhibition of cell migration by 25% in comparison to the antimir/R8 negative control group [137]. Sariah Khormaee et al evaluated the effect of covalently attaching stathmin-targeted siRNAs to a PP75 cell penetrating peptide [138].…”