Self-Assembling Betulinic Acid and Squalene Nanoparticles Enhance the Blood−Brain Barrier Reinforcing Activity of Trehalose

Abstract Trehalose, a natural disaccharide with well-established neuroprotective effects in the central nervous system (CNS), has been extensively investigated for its anti-inflammatory, protein-stabilizing, and autophagy-inducing activities. Despite these beneficial CNS effects, its potential impact on the blood−brain barrier (BBB), a key regulator of brain homeostasis and neurological health, remains poorly understood. Here, we demonstrate that trehalose reinforces the in vitro BBB by increasing transendothelial electrical resistance and reducing dextran permeability in hCMEC/D3 monolayers. These functional improvements were associated with AMPK activation and upregulation of the tight junction protein ZO-1, where findings were corroborated in vivo by increased ZO-1 expression in trehalose-treated C57BL/6J mice. Molecular docking further suggested the preferential binding of trehalose to the ZO-1 PDZ3-SH3 domain, supporting a potential mechanism for tight junction stabilization. These findings establish trehalose as a promising BBB-protective agent through complementary molecular mechanisms. Nevertheless, the therapeutic exploitation of free trehalose is hindered by its unfavorable pharmacokinetic properties. To address these limitations and improve its delivery, we engineered four self-assembling trehalose nanoparticles (Treh-NAs) based on mono- or bis-betulinic acid and mono- or bis-squalene conjugates. Nanoparticle encapsulation preserved the barrier-enhancing activity of trehalose while markedly reducing the effective dose in vitro. Among the formulations, the mono-betulinic acid nanoparticles displayed the highest endothelial permeability and the strongest barrier-reinforcing effect. Collectively, these results provide a rationale for the development of trehalose-based nanomedicines aimed at reinforcing the BBB and improving therapeutic interventions for CNS diseases associated with BBB dysfunction, in which the neuroprotective properties of trehalose may provide additional therapeutic benefit.