Glioblastoma (GBM) remains the most aggressive and lethal form of brain tumour, highlighting the urgent need for new therapeutic strategies. Gold nanorod (GNR)-mediated photothermal therapy (PTT) is a promising approach that uses near-infrared (NIR) irradiation to induce hyperthermia and selectively ablate tumour cells containing GNRs. However, improving the targeting specificity and reducing the treatment intensity are necessary for the clinical translation of this therapy to minimise off-target damage. Furthermore, achieving a uniform spatial distribution of GNRs within the tumour is essential for enhancing efficacy.

This study evaluates three strategies for functionalising the surface of GNRs: PEGylation; conjugation with Protein G; and a novel construct combining Protein G with anti-CD133 antibodies for targeted delivery to glioblastoma stem-like cells. The nanoparticles were characterised using dynamic light scattering (DLS) and zeta potential analysis to confirm successful conjugation and stability.

Spatial biology analyses were conducted using optical microscopy to visualise the anchoring of GNRs on target cells, thereby confirming the specific localisation of CD133-functionalised nanoparticles. In CT2A glioblastoma cells, CD133-GNRs exhibited the highest level of cytotoxicity (86%) under baseline conditions (4.5 W laser, 3 µg/mL), enabling a reduction in both the laser power (3 W) and the GNR dose (2 µg/mL) while maintaining effectiveness (85%).

To enhance the distribution of GNRs in tumours, macrophages were investigated as delivery vehicles due to their innate tumour-tropic behaviour. GNR-loaded macrophages were co-cultured with glioblastoma cells and their spatial migration was monitored. Upon irradiation, this strategy enhanced tumour ablation, demonstrating improved intratumoral dispersion and cell death.

These results support the use of CD133-targeted GNRs as a safer and more effective PTT modality. Furthermore, spatial analyses and intratumoral mapping confirmed the importance of localisation strategies for nanoparticles, providing valuable insights into the development of advanced nanomedicine platforms for GBM.