Design of bioengineered glioblastoma microenvironment models for the validation of innovative nanomedicines

Purpose/Objectives: The development of effective treatments for aggressive diseases like glioblastoma (GBM) is hampered by the intricate tumour microenvironment (TME). The high histological complexity of the TME, the stiff extracellular matrix (ECM), and the presence of the blood-brain barrier represent a significant barrier to effective drug delivery. To facilitate the development of more efficient therapies able to bypass these barriers, it is imperative to establish reliable in vitro models that faithfully replicate the intricate complexity of the TME. This study aims to create a robust three-dimensional model of GBM for studying the transport dynamics of drug delivery platforms based on polymer nanoparticles (NPs). This model integrates various cell types present in human GBM, biomaterials resembling the tumour ECM, and a microfluidic device to reproduce vascularization, ensuring an accurate representation of GBM's structure and composition. Methodology: Core-shell polyurethane NPs with a phospholipid coating were prepared via nanoprecipitation method. GBM spheroids, incorporating glioblastoma cells (U87 or U251), cancer-associated stem cells (GBM-8), were embedded in natural (collagen) or synthetic (VitroGel 3D) polymeric hydrogels with mechanical properties akin to the GBM matrix. Moreover, resident brain cells, like microglia (HMC3) and astrocytes (HASTR), were incorporated into the gel to investigate their role in TME. The model was employed to assess the effect of the administration of NPs for the controlled release of Bortezomib (BTZ), a proteasome inhibitor in reducing the infiltration capacity and viability of the tumor model. To verify NPs extravasation across brain capillaries, a vascular network was established by introducing GBM spheroids into a commercially available microfluidic platform featuring two lateral perfusion channels coated with human brain endothelial cells (MIMETAS OrganoPlate Graft). Angiogenic sprouting was then induced by the administration of a growth factor mix to obtain vascularization of the spheroid. Results: The GBM spheroids could replicate some important features of the tumour, such as the presence of the necrotic core and the formation of the stem cells niche (Fig.1A). The viability results on GBM spheroids after the administration of NPs showed that the treatment efficacy increased over time and with NPs concentration. BTZ and BTZ-NPs reduced tumour proliferation and infiltration in ECM gels, with efficacy dependent on cellular composition. NPs-mediated treatment exhibited lower anti-tumour efficacy compared to free BTZ probably due to the retarded drug release, but also minimized the undesired cytotoxicity on non-tumour cells. The vascular network model included dense and homogeneous vessels forming well branched sprouts, supporting spheroid vascularization (Fig. 1B). Furthermore, immunofluorescence confirmed the presence of tight junctions (Fig. 1C), replicating the barrier effect against NPs observed in vivo. Moreover, the developed model enabled the replication and investigation of microglia extravasation and tumour homing (Fig.1D), indicating its potential as a validation platform for cell-mediated transport systems. Conclusion/Significance: This innovative model represents a significant stride in replicating the human GBM TME by combining biomaterials and microfluidics. It offers a valuable tool for the preliminary validation of nanomedicines, facilitating the exploration of new materials and delivery mechanisms, such as cell-mediated drug delivery, to overcome the challenges posed by the TME in glioblastoma treatment.