Engineering of Dual-Functionalized Intranasal Nanovesicles Embedded with Thymoquinone for Targeted Modulation of the PI3K/AKT Pathway in Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, known for its invasiveness, therapy resistance, and the challenge posed by the blood-brain barrier (BBB). This study presents a novel, dual-functionalized nanovesicular system for intranasal delivery of thymoquinone (TH), encapsulated in PEGylated chitosan-coated NVs and conjugated with lactoferrin (Lf). This formulation (TH-Lf-PEG-CS-NVs) was optimized using a central composite design and exhibited high entrapment efficiency (96.28 ± 0.65%), a particle size of 129.82 ± 0.53 nm, and coating efficiency of 91.72 ± 0.22%. In vitro and ex vivo studies demonstrated superior cellular uptake (84.01 ± 3.78%) and a ∼3.16-fold increase in permeability (<i>P</i>_app: 0.57 ± 0.072 μg/cm²/min) compared to free TH. Mechanistic assays confirmed modulation of the PI3K/AKT pathway, with reduced Bcl-2 (56.91 ± 2.98%), COX-2 (31.22 ± 2.19%), and increased caspase-3 (75.9 ± 3.01%) expression. In vivo pharmacokinetics revealed a peak cerebrospinal fluid concentration (<i>C</i>_max) of 51.72 ± 1.22 μg/mL at 24 h and sustained release for 36 h. Histopathological evaluations affirmed safety and biocompatibility. This multifunctional nanocarrier facilitates targeted delivery, effective BBB penetration, and prolonged cerebral retention, offering a noninvasive, efficient strategy for GBM treatment. The findings support its potential as a translational therapeutic platform for central nervous system malignancies.