Biomimetic nanocarriers against Glioblastoma: microglia and microglia-derived vesicles as innovative delivery platforms

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults, characterized by high invasiveness, resistance to therapy, and a low median survival. Physiological barriers of the central nervous system (CNS), the blood-brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier, limit drug delivery, representing a challenge for treatment. To overcome these limitations, nanoparticles (NPs)-based delivery systems that can cross the BBB and release drugs in a controlled manner have been developed. However, this approach also presents challenges such as rapid clearance by the immune system and limited distribution within the tumor microenvironment (TME). Cell-mediated transport, exploiting the natural migration ability of immune cells, offers a promising strategy to overcome these obstacles. This study examines the use of the human microglial cell line (HMC3) as a carrier for polymer-lipid NPs loaded with the anticancer drug BASHY-bortezomib (BASHY-BTZ), evaluating their effectiveness in vitro. Two approaches were compared: direct use of microglia loaded with NPs and the use of extracellular vesicles (EVs) derived from these cells, which encapsulate the same NPs. The NPs, consisting of a polyurethane core and a lipid shell, showed uniform size (150 nm), low polydispersity (PDI < 0.2), and good encapsulation efficiency (35%). Moreover, HMC3 cells internalized NPs in a concentration and time-dependent manner: at high concentrations (0.5 to 1 mg/mL), over 80% uptake occurred within 1 hour and remained stable up to 24 hours, while lower concentrations (e.g., 0.1 mg/mL) showed a gradual increase over time. Migration experiments in collagen hydrogel confirmed the selective tropism of microglia toward GBM. HMC3 cells demonstrated a strong ability to migrate toward brain tumor cells (U87 and U251), but not toward healthy cells or other tumor types. Similarly, in a commercial microfluidic platform, microglia cells displayed the tendency to migrate towards tumor cells (traveling about 200 µm in 7 days), penetrating across in vitro vessels. HMC3 cells loaded with BASHY-BTZ-NPs significantly reduced U87 cell viability in both 2D cultures and 3D spheroids, with a response proportional to the administered dose. NPs-loaded EVs were isolated from HMC3 cells through ATP stimulation and showed appropriate physicochemical properties (hydrodynamic diameter around 260 nm, PDI < 0.25 and negative zeta potential) for drug delivery applications. U87 tumor cells effectively internalized NPs-loaded EVs in both 2D monolayers and 3D spheroid models, with good penetration observed even in the deeper spheroid regions. These results were also supported by quantitative cytofluorimetry analysis, showing over 60% uptake of NPs-loaded EVs. Following administration of EVs containing BASHY-BTZ-NPs to 2D and 3D tumor models, a significant antitumoral effect was observed, with a reduction of U87 spheroid viability up to 53% after 72 hours. Overall, these results highlight the potential of microglia, both as cellular carriers and as sources of EVs, for targeted and effective drug delivery to GBM.