Asymptotic r 2 Enhancement in Formulation‐Optimized Lipid‐Iron Oxide Nanoparticles for Assessing Glioblastoma Heterogeneity at Ultrahigh‐Field MRI

Glioblastoma (GBM) remains a major challenge for nanomedicine because heterogeneous tumor vascularization and the blood–brain barrier cause variable nanoparticle accumulation. Here, we developed lipid‐coated superparamagnetic iron oxide nanoparticles (LSPIONs) as MRI‐visible lipid nanocarriers and established a formulation‐based strategy to control their transverse relaxivity (r 2 ). Hydrophobic iron oxide nanoparticles (IO@OA) were incorporated into a DSPC/cholesterol/DSPE‐PEG lipid matrix via solvent displacement, enabling systematic control of nanoparticle size, polydispersity, and SPION loading, with r 2 values ranging from 13 to 418 m m − 1 s − 1 at 9.4 T. The relaxivity showed an asymptotic dependence on nanoparticle size (54–184 nm) and estimated iron oxide nanoparticles per lipid nanoparticle (237–9284), revealing a saturation regime. The optimized formulation, obtained at a lipid‐to‐IO@OA molar ratio of 8.3 (D H = 98 nm, PDI = 0.14), was evaluated in a syngeneic GL261‐GFP GBM mouse model. Intravenous administration induced an initial reduction in GBM‐associated MRI signal, with detectable tumor retention at 24 h in tumors assessed at 1 and 2 weeks after inoculation. Intravital multiphoton microscopy further confirmed LSPION extravasation and GBM accumulation. These results establish formulation control of r 2 as a key design strategy for MRI‐visible lipid nanocarriers and provide an experimental framework for lipid‐based nanoprobes for GBM imaging.