Peptide-Based Nanocomplexes Enable Transferrin-Mediated Uptake and p53-Driven Antitumor Activity in 2D and 3D Glioblastoma Models

Glioblastoma (GB), the most prevalent and aggressive brain tumor, remains one of the most lethal challenges in modern oncology. Standard therapy remains largely ineffective, mainly due to limited therapeutic penetration across the blood–brain barrier (BBB) and adaptive tumor resistance. Consequently, there is an urgent need for innovative strategies to enhance therapeutic precision and efficacy. To address these limitations, we engineered a targeted peptide-based co-delivery system using the WRAP5 cell-penetrating peptide functionalized with a transferrin receptor (TfR)-targeting T7 peptide (sequence: HAIYPRH), enabling simultaneous delivery of temozolomide (TMZ) and a p53-encoding plasmid DNA. The resulting peptide-based TMZ/p53 nanocomplexes exhibited favorable physicochemical properties, enhanced TfR-mediated cellular uptake, and targeted antitumor activity mediated through p53-induced apoptosis in two-dimensional (2D) U87 MG cell cultures. To better reproduce the structural and cellular complexity of the tumor microenvironment, a three-dimensional U87 MG spheroid model was established and optimized using a Design of Experiments (DoE) approach to improve reproducibility and physiological relevance. The developed WRAP5-based nanocomplexes induced a significant dose-dependent inhibition of growth and morphological alterations in the U87 MG spheroid model, accompanied by deep penetration and cell death throughout the spheroid. Collectively, these findings highlight the potential of this targeted and tailored nanosystem to enhance cellular transfection, enable drug/gene co-delivery, restore p53 function, and promote apoptosis, representing a promising therapeutic strategy for GB treatment.