Presentation Information
[4FMBS-04]Engineering a Thermoresponsive “Thermal Switch” in Human Ferritin Nanocages for Rapid, Non-Destructive Drug Encapsulation
○Hsiao-Ching Su1, Chien-Yi Chang1, Chiun-Wei Huang2, Feng-Ting Huang1 (1. Department of Biochemical Science and Technology, National Taiwan University, Taipei (Taiwan), 2. Department of Medical Research and Development, Linkou Chang Gung Memorial Hospital, Taoyuan (Taiwan))
Keywords:
Ferritin nanocages,Thermoresponsive channels,Protein engineering,Rational design,Drug encapsulation,Glioblastoma therapy
[Purpose] Human ferritin heavy chain (HFn) nanocages are premier candidates for glioblastoma therapy due to their innate ability to traverse the blood-brain barrier via TfR1. However, conventional drug loading typically necessitates harsh pH-induced disassembly/reassembly or prolonged thermal stress (4-6 hours), both of which can compromise cargo stability and protein monodispersity. This study aimed to engineer a “thermal switch” into the HFn architecture to enable rapid, high-efficiency drug encapsulation under mild, non-destructive conditions.
[Method] Using structure-guided design, we targeted the two-fold symmetry interface—the primary gatekeeper for molecular transport in HFn. AlphaFold3 modeling and molecular dynamics (MD) simulations identified a critical hydrogen-bond network (R43-D45) that restricts interfacial breathing. We performed site-directed mutagenesis (D44A) to disrupt these constraints and increase local flexibility. Loading kinetics and capacity were evaluated using doxorubicin (DOX) and RSL3, with structural integrity verified via circular dichroism and native PAGE. Finally, the biological efficacy and glioblastoma-targeting potential of the engineered nanocages were rigorously assessed through both in vitro assays and in vivo therapeutic models using RGD-functionalized variants.
[Results] MD simulations revealed that the D44A mutation creates a temperature-dependent “loosening” of the inter-subunit channels. Experimentally, the D44A variant achieved a loading capacity of ~94 DOX molecules per nanocage in only 30 minutes at 60°C—a significant acceleration compared to wild-type HFn, which showed minimal uptake under identical conditions. Crucially, the engineered nanocages remained monodisperse and retained pH-responsive release. In vivo, RGD-functionalized D44A nanocages demonstrated superior tumor suppression and reduced systemic toxicity compared to free drug formulations.
[Consideration] These results indicate that tuning interfacial interactions can effectively regulate thermoresponsive channel dynamics without compromising nanocage stability. Compared to conventional approaches, this strategy provides a mild, efficient, and broadly applicable platform for loading diverse cargos, ranging from hydrophilic to hydrophobic therapeutics.
[Conclusion] We have successfully transformed the HFn nanocage into a thermoresponsive nanocarrier by fine-tuning interfacial dynamics. By substituting a single amino acid, we achieved a significant increase in loading efficiency and a ten-fold reduction in processing time without resorting to denaturing conditions. This rational engineering approach provides a robust framework for developing next-generation protein-based delivery platforms.
[Method] Using structure-guided design, we targeted the two-fold symmetry interface—the primary gatekeeper for molecular transport in HFn. AlphaFold3 modeling and molecular dynamics (MD) simulations identified a critical hydrogen-bond network (R43-D45) that restricts interfacial breathing. We performed site-directed mutagenesis (D44A) to disrupt these constraints and increase local flexibility. Loading kinetics and capacity were evaluated using doxorubicin (DOX) and RSL3, with structural integrity verified via circular dichroism and native PAGE. Finally, the biological efficacy and glioblastoma-targeting potential of the engineered nanocages were rigorously assessed through both in vitro assays and in vivo therapeutic models using RGD-functionalized variants.
[Results] MD simulations revealed that the D44A mutation creates a temperature-dependent “loosening” of the inter-subunit channels. Experimentally, the D44A variant achieved a loading capacity of ~94 DOX molecules per nanocage in only 30 minutes at 60°C—a significant acceleration compared to wild-type HFn, which showed minimal uptake under identical conditions. Crucially, the engineered nanocages remained monodisperse and retained pH-responsive release. In vivo, RGD-functionalized D44A nanocages demonstrated superior tumor suppression and reduced systemic toxicity compared to free drug formulations.
[Consideration] These results indicate that tuning interfacial interactions can effectively regulate thermoresponsive channel dynamics without compromising nanocage stability. Compared to conventional approaches, this strategy provides a mild, efficient, and broadly applicable platform for loading diverse cargos, ranging from hydrophilic to hydrophobic therapeutics.
[Conclusion] We have successfully transformed the HFn nanocage into a thermoresponsive nanocarrier by fine-tuning interfacial dynamics. By substituting a single amino acid, we achieved a significant increase in loading efficiency and a ten-fold reduction in processing time without resorting to denaturing conditions. This rational engineering approach provides a robust framework for developing next-generation protein-based delivery platforms.
Comment
To browse or post comments, you must log in.Log in