Nose-to-Brain Nano-in-Microparticles for Brain Tumours with Targeted Microfluidically Continuous Manufactured Peptide Nanofibers

Glioblastoma multiforme (GBM) patients have a median survival of 15 months after surgery, radiotherapy and temozolomide treatment. Gonadotrophin releasing hormone (GnRH) receptors 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. Here, we aim to develop continuous microfluidic manufacture of these nanomedicines and their nano-in-microparticles towards nose-to-brain non-invasive targeted therapies for GBM.
TPGnRH was synthesised using an orthogonically protected SPSS protocol. A staggered herringbone micromixer [microchannel: 21 um × 56 um, herringbone grooves: 4 um × 20 um] was used and aqueous dispersions of TPGnRH and paclitaxel solutions in isopropyl alcohol were mixed at flow rate ratios (FRRs) ranging between 1:1 and 1:8 v/v (0.819 mL/min). Computational fluid dynamics (CFD) studies (Star-CCM+) were used in conjunction with experimental data to optimise the FRR. Critical aggregation concentration, paclitaxel loading, size, zeta-potential and circular dichroism studies were undertaken. Nano-in-microparticles (NIM) were prepared with pullulan polymers and spray-drying and TPGnRH and NIM permeability across lamb nasal mucosa and RPMI2650 cell monolayers cultured in air liquid and liquid-liquid conditions is assessed.
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, TEM) were observed similar to solvent evaporation. Low FRR ratios (1:1) showed the presence of spherical aggregates (120 ± 25 nm ), while midrange FRR indicated predominantly long axial particles. Peptide aggregates were stabilised via poly(proline) type Il helixes as TPGnRH and the method of manufacture did not impact on the secondary structure of the amphiphile aggregates or in vitro efficacy in U87MG cells. The encapsulation efficiency was 85 ± 15% compared to 97±5% with batch processes. Microfluidically prepared nanofibers and NIM were permeable across lamb nasal mucosa and RPMI2650 monolayers.
Current approach demonstrates the microfluidic manufacture of long axial nanofibers and the continuous manufacture of nano-in-microparticulate targeted therapies for brain diseases and cancers.