Computer-aided drug design of SP94 peptide-functionalized human H-chain ferritin for targeted doxorubicin delivery

Human H chain ferritin (HFtn) is an established nanocarrier for antitumor drug delivery, with peptide modifications enhancing its tumor-targeting specificity. The linker connecting targeting peptides to HFtn is critical for maintaining functionality, yet traditional linker screening methods are labor-intensive and time-consuming. This study utilized computer-aided drug design (CADD) to screen linkers for fusing the SP94 peptide, which targets Glucose-Regulated Protein 78 (GRP78) overexpressed in hepatocellular carcinoma cells, to HFtn. Molecular dynamics simulations and binding free energy analyses identified the flexible (GGGGS)₂ linker as optimal for the SP94-HFtn fusion protein. The engineered SP94-(GGGGS)₂-HFtn was expressed and loaded with doxorubicin (DOX) using a thermal treatment method, which outperformed pH and urea-based methods in drug loading capacity and protein recovery. In vitro evaluations demonstrated that SP94-(GGGGS)₂-HFtn-DOX nanoparticles exhibited superior cellular uptake, enhanced cytotoxicity against HepG2 tumor cells, and improved biosafety in normal cells compared to HFtn-DOX nanoparticles. These findings highlight the efficacy of CADD-guided linker selection in developing targeted ferritin-based nanocarriers, with the thermal treatment method offering a robust strategy for efficient drug loading. This approach provides a framework for designing precision nanomedicines with enhanced therapeutic efficacy and safety.