Design of bioresponsive nanoparticles for the local treatment of glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor. The standard of care for GBM has remained unchanged since 2005 and consists of surgical resection, radiotherapy, chemotherapy with temozolomide combined with corticosteroids; despite this, the median survival rate for patients is 14-16 months. The limitations associated with treating this unmet medical need stem from the presence of the blood-brain barrier (BBB), the heterogeneity of the tumor, mechanisms of drug resistance, and the immunosuppressive tumor immune microenvironment (TIME). This study proposes the development of polymeric nanoparticles (NPs) intended for convection-enhanced delivery (CED) in GBM. Specifically, those NPs were designed to be bioresponsive to specific intrinsic GBM stimuli: a) the acidic pH inside the bulk of tumor cells and inside the cells themselves, and b) the oxidative stress caused by reactive oxygen species (ROS), and the intracellular glutathione of the TIME. Owing to this bioresponsiveness, the NPs could selectively release their therapeutic payload within tumor cells, inducing cytotoxic effects exclusively in malignant cells while sparing healthy tissue. Starting from Hyaluronic Acid and Polylysine, NPs were covalently crosslinked using a microfluidic approach, using tailored bioresponsive linking chemistries. Different flow rates were explored to yield NPs with different features. The chemical identity of all the polymers and linkers was ensured using NMR and ESI-mass Spectrometry. The properties of the NPs (size, PDI, and zeta potential) were evaluated using dynamic light scattering (DLS). It was demonstrated that the non-bioresponsive NPs possess the desired properties for penetration into the brain parenchyma: a size ≤ 100 nm with PDI ≤ 0.2, and a negative Z potential. The pH-responsive NPs were engineered using two distinct linkers (hydrazine-levulinic acid-tetrazine and oxalyldihydrazide-levulinic acid-tetrazine), and their disassembly under acidic conditions was evaluated. A marked increase in particle size was observed, from 133 nm at pH 7.4 to 213 nm at pH 5.5. Likewise, the ROS-responsive NPs were prepared using a linker tioketal-tetrazine, demonstrated stimulus-dependent behavior, exhibiting a size enlargement from 279 nm to 748 nm in cerebrospinal fluid (CSF) at pH 7.4 upon exposure to H₂O₂. Collectively, these findings underscore the critical role of thorough linker characterization in promoting in vitro assembly/disassembly kinetics for brain-penetrating NPs triggered by endogenous stimuli. Further steps of linker optimization are required to obtain suitable bioresponsive NPs for in vivo application.