Design of nanomedicines for targeted delivery and monitoring of chemotherapeutics in Glioblastoma treatment

Glioblastoma multiforme (GBM) is a grade IV glioma originating from astrocytic glial cells and one of the deadliest tumors of the central nervous system (CNS), with an expected survival below 15 months. Standard therapies combining surgical resection, radiotherapy, and chemotherapy with temozolomide are often ineffective, as GBM invasiveness hampers the complete tumor removal, promoting recurrence. Furthermore, the complexity and heterogeneity of the tumor microenvironment (TME) hinders drug efficacy, while the presence of the blood-brain barrier (BBB) prevents the passage into the nervous vascular compartment. Recently, new drug delivery systems (DDS) have been designed to facilitate the accumulation of small molecules in the tumor and improve targeted treatments. This study aims to develop nanoparticles (NPs) to encapsulate Bortezomib (BTZ), a proteasome inhibitor whose therapeutic application is limited by the high off-target toxicity and the low accumulation in the GBM mass. Core-shell BTZ-loaded NPs with a uniform size around 150 nm and a low polydispersity index (< 0.2) were produced by nanoprecipitation, providing an encapsulation efficiency (EE) of 10%. The therapeutic efficacy of the NPs was evaluated in vitro using two-dimensional and three-dimensional (spheroids) models of different human GBM cell lines (U87, U251), and compared with direct administration of BTZ. U87 and U251 rapidly internalized the NPs, with an uptake above 70% after 2 h of incubation with 0.1 mg/mL NPs. The efficacy of the treatments increased with concentration and incubation time. After 72 h, BTZ-NPs (500 nM) reduce cell viability by up to 20%, with no significant difference compared to free BTZ in both 2D and 3D models. Other tumor properties, such as the regenerative capacity of cell layers and the growth of necrotic regions within spheroids, were assessed after treatment, confirming that NPs elicit a therapeutic response on both GBM cell lines, although the effect was less pronounced than for free BTZ. Additionally, a fluorescent conjugate of BTZ, i.e., BASHY-BTZ (University of Lisbon), was assessed (in free form and encapsulated inside NPs) to enable intracellular drug tracking, while preserving the therapeutic effect of BTZ. The use of BASHY BTZ increased the EE up to 40% without altering the physical-chemical characteristics of the NPs and enabling the simultaneous monitoring of the drug and the NPs inside GBM cells through microscopy and cytofluorimetry. In vitro results confirmed that BASHY BTZ and BASHY-BTZ-NPs presented significant antitumoral properties in both 2D and 3D models, reducing GBM cell viability by up to 33% after 72 hours of treatment (500 nM). Lastly, the NPs were functionalized with transferrin (Tfr), a protein that can enhance particle penetration across the BBB and GBM targeting. The functionalization with Tfr increased NPs internalization within 24 hours on both U87 and U251, without compromising the drug action against GBM cells, confirming its potential as an active targeting strategy. Overall, these results highlight the potential of our NPs as systems for the controlled and effective delivery of small molecules, capable of overcoming the limitations of conventional GBM therapies. These nanomedicines could constitute a unique and versatile platform for cancer treatment, through their active targeting and the possibility of improving imaging and monitoring in preclinical in vitro and in vivo models.