Microfluidic manufacture of peptide nanofibers for targeting glioblastoma multiforme

Glioblastoma multiforme (GBM) affects 8 in 100,000 in the UK and patients have a median survival of 15 months after surgery, radiotherapy and temozolomide treatment. Gonadotrophin releasing hormone (GnRH) are overexpressed in GBM patients, but GnRH is unable to cross the blood-brain barrier and is enzymatically unstable with a half-life of 3 minutes. We have shown that GnRH amphiphiles (TPGnRH) assemble into stable nanofibers that can bind GnRH GPCR and elicit an antiproliferative response at low micromolar concentrations causing G2/M phase arrest, while being able to cross the blood-brain barrier and target loaded cytotoxics to GBM cells. Solvent evaporation batch processes were used in manufacture of these peptide nanomedicines. Here, we aim to assess the continuous microfluidic manufacture of these peptide nanomedicines towards achieving a nose-to-brain non-invasive therapy for GBM.
TPGnRH was synthesised using an orthogonically protected SPSS protocol. A staggered herringbone micromixer [microchannel: 21 μm (height) × 56 μm (width), herringbone grooves: 4 μm (depth) × 20 μm (width)] was used and aqueous dispersions of TPGnRH and paclitaxel (anticancer drug) solutions in isopropyl alcohol were mixed at flow rate ratios (FRRs) ranging between 1:1 and 1:8 v/v (0.819mL/min). Computational fluid dynamics (CFD) studies (Star-CCM+) were used in conjunction with experimental data to optimise the required FRR. Critical aggregation concentration, paclitaxel loading, size, zeta-potential and circular dichroism studies were undertaken for both batch and microfluidic nanomedicines.
Based on the mass fraction of each mixed fluids, near-perfect mixing was achieved earlier within the chip for higher FRRs (1:8) and lower total flow rates. At FRR of 1:8, long axial nanofibers (450 ± 282 and 55 ± 30 nm) were observed using transmission electron microscopy similar to solvent evaporation. Low FRR ratios (1:1) demonstrated the presence of spherical aggregates (120 ± 25 nm in diameter), while mid-range FRR indicated predominantly long axial particles. All three peptide aggregates were stabilised via poly(proline) type II helixes as TPGnRH and the method of manufacture did not impact on the secondary structure of the amphiphile aggregates. The encapsulation efficiency was 80 ± 15% compared to 97±5% with batch processes. Larger scale studies and comparison of their in vitro efficacy in U87MG cells are currently underway. Eluted formulations will then be converted into nano-in-microparticles via atomisation for nose-to-brain delivery application.