Bioengineered Dual‐Targeting Protein Nanocage for Stereoscopical Loading of Synergistic Hydrophilic/Hydrophobic Drugs to Enhance Anticancer Efficacy

A biocompatible and modifiable protein nanocarrier is a promising candidate for tumor targeted drug delivery. However, it is challenging to effectively load hydrophobic drugs, not to mention to upload both hydrophilic and hydrophobic drugs on one protein nanocarrier. Here, an amphiphilic multi‐drug loading protein nanocage (Am‐PNCage) is presented which is generated by replacing the fifth helix of human H‐ferritin (HFn) subunit with a functional motif composed of hydrophobic–hydrophilic‐RGD peptides. The Am‐PNCage possesses a dual targeting property resulting from the intrinsic CD71 targeting ability of HFn and the integrin α vβ3 targeting ability of displayed RGD peptides. Through the hydrophilic drug entry channel in the protein nanocage and hydrophobic peptides displayed on the outer surface, amphiphilic epirubicin (132)/camptothecin (50) are stereoscopically loaded into the inner cavity/outer protein shell, respectively, for one Am‐PNCage, exhibiting cascade drug release pattern. The dual‐targeted Am‐PNCage promotes the loaded drugs penetrating various 3D tumor models in vitro, as well as traversing the brain blood barrier and accumulating in brain tumors in vivo. Moreover, the drug loaded Am‐PNCage shows reduced side effects and significantly enhances synergistic efficacy against brain tumor, metastatic liver cancers, and drug resistant breast tumor. Thus, the Am‐PNCage represents a novel promising protein nanocarrier for targeted combination chemotherapy. The dual tumor‐targeting and amphiphilic nanocarrier Am‐PNCage extends the half‐life of loaded drugs, reduces their side effects, promotes their binding, and penetration into tumors. Together with a synergistic cascade drug release, these improvements lead to a significantly enhanced therapeutic effect. Thus, the Am‐PNCage represents a novel promising platform for co‐loading of synergistic hydrophilic and hydrophobic drug pairs and targeted combination chemotherapy.