Trehalose-delivering nanoparticles improve the blood-brain barrier tightness

The brain is a privileged immune organ whose protection and homeostasis are ensured by the blood-brain barrier (BBB), which is a dynamic interface between the brain and the bloodstream. Variations and impairments in the structure and function of the BBB have been reported for different brain pathologies, brain cancer included. Therefore, strategies to protect BBB integrity could improve its functionality, mitigating disease progression. Trehalose is a natural glucose disaccharide with multiple pharmacological activities, including the induction of autophagy, inhibition of protein aggregation, and neuroprotection. However, little information is known about its effect on the BBB. To this purpose, we investigated the potential beneficial effect of trehalose on the bioelectrical, structural, and functional properties of the BBB using human brain capillary endothelial cells (hCMEC/D3) seeded in a transwell system. Results showed that trehalose improves barrier performance and functions, leading to increased TEER and reduced paracellular permeability to fluorescent probes. Moreover, the expression of the tight junction proteins increased after trehalose treatment, demonstrating the positive impact of the bioactive molecule. However, trehalose shows poor permeability through biological membranes and a short half-life. Therefore, lipidic trehalose nanoparticles with four different conjugates (mono-squalene, bis-squalene, mono-betulinic acid, and bis-betulinic acid) were synthesized, assembled, and characterized (with DLS, ζ-potential, and TEM). Results showed that the formulation of trehalose in nanoparticles improved its ability to cross the BBB and increased its stability and half-life. Such activity was observed at much lower doses, which is useful for protecting the BBB. In addition, trehalose will be investigated as a therapeutic candidate for glioblastoma therapy. Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour, associated with a median long-term survival of 15 months after diagnosis. The standard treatment for patients diagnosed with GBM is maximal surgical resection followed by radiotherapy. However, significant tumour heterogeneity and unsuccessful recurrence management highlight the urgent need for improved therapeutic strategies. The literature has reported that the mechanisms of GBM progression and treatment resistance involve GBM interacting with the brain microenvironment, including astrocytes. Taking into consideration the above-mentioned complexity of the brain microenvironment in pathological conditions, in vivo models are particularly useful in the development of new therapeutic strategies. However, the main limitation is that the growth of GBM in animal models requires the use of immunocompromised mice, which prevents the translation of results to human tissue and raises ethical concerns. Therefore, other more realistic systems recapitulating human GBM diversity will improve understanding of molecular heterogeneity and therapeutic efficacy. In line with the principles of the 3Rs, these systems will avoid the use of animals unless deemed necessary. Recently, 3D models have become a valuable tool for studying brain tumours, as they recapitulate the complexity of the human tumour microenvironment. In this context we are developing a sustainable and reproducible 3D glioblastoma model for testing smart nanosystems targeting brain tumours. The model is developed in collaboration with Real Research SA that provides innovative technologies for experimental standardization using LifeGel technology. Validation for drug and nanosystem screening will be conducted at DIPC research center in San Sebastián. Preliminary results using U87 glioblastoma cells demonstrate successful adaptation to LifeGel technology and 3D structure formation with optimized settings. This will establish a robust platform for therapeutic development, reduce dependency on animal models, and support ethical, translatable research.