Multivalent peptides for blood-brain barrier translocation, cell penetration and microRNA-21 inhibition in glioblastoma.

Glioblastoma (GBM) is an aggressive brain cancer for which the prognosis remains poor despite the advances in our understanding of the complex heterogeneity of the disease. Therapeutic access to GBM tumours requires drugs to cross the blood-brain barrier (BBB), a physiological obstacle that restricts the passage of compounds from systemic circulation into the brain. Blood-brain barrier peptide shuttles (BBBpS), which are a subclass of cell penetrating peptides (CPPs), represent a promising approach for transferring large cargos across the BBB, especially cargos such as peptides or antisense oligonucleotides against RNA targets. Such RNA targets include microRNAs (miRNA), of which several have emerged as candidate drug targets in GBM. MicroRNA-21 is one such miRNA implicated in GBM, and although peptide-based miR-21 inhibition has been reported, delivery of peptide-based inhibitors of miR-21 across the BBB has received limited attention. Here, we selected a BBB-penetrating peptide, SYPGWSW, for conjugation to a peptide-based inihibitor of miRNA processing. We show that SYPGWSW penetrates GBM cell lines and its function depends critically on the properties of aromatic residues Tyr and Trp but not on Pro residues. Further, subsitution of Tyr with unnatural amino acids cyclohexylalanine or 1-naphthylalanine enhanced the internalisaton of the parent peptide by 2 to 4 fold. Mechanistically, the higher level of uptake was associated with enhanced stability of the peptide, which appeared to be partly associated with binding to human serum albumin. Finally we showed that a peptide inhibitor of miR-21 processing conjugated to SYPGWSW traversed an in vitro BBB model and enabled the functional silencing of miR-21-5p in GBM cells. Together, these findings pave the way for peptide conjugates as novel therapeutics for silencing miRNA expression in brain cancer.