iRGD-Functionalized Graphene–Silver Nanohybrids for Enhanced Blood–Brain Barrier Transport and Tumor Penetration in Glioma Models

Efficient drug delivery across both the tumor microenvironment and the blood-brain barrier (BBB) remains a major challenge in glioblastoma therapy, where dense cellular architecture and endothelial restriction severely limit treatment efficacy. Here, we report the rational design of a multifunctional nanoplatform, Dox[iRGD-GO@AgNP], integrating graphene oxide (GO), silver nanoparticles (AgNPs), and a cyclic iRGD tumor-penetrating peptide to enhance doxorubicin (Dox) transport, tumor penetration, and intracellular delivery. Comprehensive physicochemical characterization confirmed successful synthesis, surface functionalization, colloidal stability, and efficient drug loading. In two-dimensional C6 glioma cell monolayers, cyclic iRGD-functionalized nanohybrids demonstrated significantly enhanced intracellular Dox accumulation compared to free drug and nontargeted controls. These effects were further amplified in three-dimensional glioma spheroids, where Dox[iRGD-GO@AgNP] achieved deep tumor penetration (>80 µm) and more homogeneous intratumoral distribution. Enhanced delivery translated into superior therapeutic efficacy, as evidenced by reduced cell viability, increased membrane damage, upregulation of apoptosis-related markers (p53 and caspase-3 &9), and pronounced morphological disruption confirmed by fluorescence imaging and electron microscopy. To evaluate endothelial transport, a Transwell-based bEnd.3 BBB model was established and validated by transendothelial electrical resistance measurements and tight junction imaging. Cyclic iRGD-functionalized nanohybrids exhibited significantly enhanced transendothelial transport while preserving BBB integrity, indicating ligand-mediated transport rather than nonspecific barrier disruption. Importantly, improved BBB traversal resulted in increased drug accumulation in downstream glioma cells and spheroids. These composites showed anti-angiogenic properties in ovo CAM model. Overall, this study integrates rational nanocomposite design with a hierarchical in vitro evaluation framework spanning material characterization, tumor-relevant 2D and 3D models, and BBB transport. The Dox[iRGD-GO@AgNP] platform addresses key delivery and efficacy barriers in glioblastoma and provides a translationally relevant strategy for the development of BBB-penetrating nanotherapeutics.