Peptide nanofibers: targeted therapies for glioblastoma multiforme?

Glioblastoma multiforme (GBM) is a malignant brain tumor with a two-year survival below 30%, even with the current advances in radio- and chemotherapy. Tumor heterogeneity, poor drug selectivity and inability of cytotoxics to cross the blood-brain barrier (BBB) remain challenges that need to be overcome for effective treat-ment. GBM biopsies have indicated the overexpression of G-protein coupled recep-tors (GPCRs) that when activated by neuropeptide agonists result in an antiprolifer-ative effect in GBM cells [1,2]. However, translation of these neuropeptides into novel therapies for the treatment of GBM is frustrated by their short in vivo half-life (<5 minutes) and inability to cross the BBB. Here, we present a novel strategy com-prised by the lipidisation of the neuropeptide agonists which results in peptide am-phiphiles able to self-assemble into nanofibers that can entrap brain impermeable drugs, possesses an enhanced stability to enzymatic degradation, permeate across an all human in vitro BBB model and are able to target the GPCRs on GBM cells re-sulting in a significant antiproliferative effect. Peptide amphiphiles were synthesized using solid-phase peptide synthesis. Their self-assembly was studied using pyrene, thioflavin T, circular dichroism and trans-mission electron microscopy (TEM) experiments. Self-assembled nanofibers were loaded with brain impermeable cytotoxic drugs such as paclitaxel and their stability was studied in biological media (plasma, brain, liver and tumor cell lysates)]. and permeation across an all human in vitro BBB transwell model were studied, and the synergistic antiproliferative effect of GnRH analogue was evaluated on GBM cells, U-87 MG. The specificity of the peptide amphiphile to the GPCR was evaluated us-ing single-molecule force spectroscopy (SMFS). Peptide amphiphiles were able to self-assemble into stable nanofibers at concentra-tions above 189 M able to load high amounts of paclitaxel (>1.8mg, > 40-fold its aqueous solubility) that can be translatable preclinically. Both unloaded- and paclitaxel loaded-nanofibers showed superior stability compared to the parent neu-ropeptide in the presence of plasma, brain, liver and GBM cells homogenates(>6-fold). Using a GPCR positive GBM cell line such as U-87 MG, the lipidised peptide elicited a strong antiproliferative effect (IC50: 5.06 M) resulting in a cell cycle arrest at G2/M and loading of paclitaxel (1nM) resulted in a synergistic effect evidenced by a decrease in cell survival by 32%. Peptide amphiphiles counteracted the forskolin-induced increase of intracellular cAMP levels supporting the GPCR link to a GI pro-tein which mediates the antiproliferative effect on GBM through the inhibition of cAMP accumulation. Confocal studies confirmed the internalisation of the peptide, while SMFS studies confirmed the specific binding of the nanofibers to GPCR on U-87MG cell surface with equivalent binding probability to endogenous neuropep-tide agonists and increased residence time. Texas Red labelled nanofibers perme-ate across an in vitro BBB model enabling the permeation of paclitaxel (Papp: 8.45 x 10-6 cm/s). Thus, described peptide nanofibers are a novel targeted nanomedicine for GBM therapy able to be clinically translated. In vivo proof of concept and phar-macokinetics studies are under way.