Structure-guided rational design of ferritin nanocages unlocks thermoresponsive channels for accelerated drug...

Human ferritin heavy chain (HFn) nanocages are attractive biological macromolecular carriers for glioblastoma therapy owing to their intrinsic biocompatibility, well-defined cage-like architecture, and transferrin receptor 1 (TfR1)-mediated blood-brain barrier penetration. HFn nanocages possess intersubunit channels that regulate molecular transport across the protein shell. However, efficient cargo loading typically requires prolonged heating (4-6 h) at elevated temperatures, limiting practical utility. Here, we report a rational, structure-guided design strategy to modulate the thermoresponsive channel located at the two-fold interface. Computational modeling with AlphaFold3 identified residues R43-D45 as key stabilizers of a hydrogen-bond network that constrains local interfacial flexibility. Substituting Asp44 with alanine (D44A) disrupted this network, increasing interfacial flexibility while preserving overall nanocage integrity. Molecular dynamics simulations revealed temperature-dependent loosening of the channel-surrounding two-fold interface, facilitating transient channel expansion for cargo entry. Consistent with these predictions, the D44A variant exhibited significantly accelerated encapsulation kinetics, achieving a loading capacity of ∼94 doxorubicin molecules per nanocage in just 30 min at 60 °C-conditions where wild-type HFn showed significantly lower uptake. Crucially, this modification was achieved while maintaining robust structural stability and pH-responsive release. Further functionalization with an RGD peptide enhanced glioblastoma-targeted cellular uptake, and DOX-loaded RGD-D44A nanocages demonstrated potent antitumor efficacy in vivo. Collectively, these findings establish a rational mutation-based approach to fine-tune channel-adjacent interfacial dynamics, providing a generalizable framework for thermoresponsive macromolecular carrier design.