A Modular Platform for Enhanced Drug Delivery to Glioblastoma Using Targeted Multidomain Protein Assemblies

Protein-based nanocarriers bear highly desirable properties such as biodegradability and the ability to facilitate passage through biological barriers such as the blood-brain-barrier. Using modular protein engineering, we develop a strategy for iteratively improving the delivery efficacy of hydrophobic small molecules for the treatment of glioblastoma multiforme (GBM). By increasing the multiplicity m of the coiled-coil and RGD peptide targeting regions from 1 to 2, we can increase both the hydrodynamic micellar size and drug loading capacity of the targeted multidomain protein assembly (TMPA) relative to its predecessor thermoresponsive assembled protein (TRAP). An upper limit of m is likely determined by steric interactions. TMPA shows a 1.7-fold increase in doxorubicin (Dox) encapsulation compared to TRAP and demonstrates a 1.3-fold improvement in uptake by U87 human GBM cells. Near-infrared (NIR) dye-labelled TMPA (NIR-TMPA) is intravenously administered to mice orthotopically implanted with GBM cells and to control mice. Pharmacokinetic analysis using a 2-compartment pharmacokinetic model reveals a significantly prolonged distribution-phase (short-phase) half-life in tumor-bearing mice compared to control, while the elimination-phase (slow-phase) half-life remains comparable between groups. This suggests altered early-phase kinetics likely due to tumor-associated sequestration or retention. The resulting increased area under the concentration-time curve (AUC) in tumor-bearing mice supports enhanced accumulation or slower clearance. Ex vivo fluorescence imaging of organs and 3D reconstructions of whole mouse heads further corroboratesa preferential localization of NIR-TMPA in tumor regions. These findings highlight the potential of TMPA and its future derivatives for targeted GBM therapy.