Ferritin, one of the most investigated protein nanocages, is considered as a promising drug carrier because of its advantageous stability and safety. However, its short half-life and undesirable tumor targeting ability has limited its usage in tumor treatment. In this work, two types of functional peptides, half-life extension peptide PAS, and tumor targeting peptide RGDK (Arg-Gly-Asp-Lys), are inserted to human heavy-chain ferritin (HFn) at C-terminal through flexible linkers with two distinct enzyme cleavable sites. Structural characterizations show both HFn and engineered HFns can assemble into nanoparticles but with different apparent hydrodynamic volumes and molecular weights. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase of growth inhibition against 4T1 cell line in vitro. Pharmacokinetic study in vivo demonstrates PAS peptides extended ferritin half-life about 4.9 times in Sprague Dawley rats. RGDK peptides greatly enhanced drug accumulation in the tumor site rather than in other organs in biodistribution analysis. Drug loaded PAS-RGDK functionalized HFns curbed tumor growth with significantly greater efficacies in comparison with drug loaded HFn.

Sorafenib, the first‐line agent for treatment of advanced hepatocellular carcinoma (HCC), improves median overall survival by approximately 3 months. In the present study, we investigated whether sorafenib combined with cucurbitacin B (CuB), a natural tetracyclic triterpenoid isolated from Cucurbitaceae, exerts enhanced antitumor effects against HCC. Cell viability and colony formation ability were detected by cell‐counting kit‐8 and colony formation assays. Cell cycle and apoptosis were analyzed by flow cytometry. Protein expression was detected by western blotting. HepG2 xenografts in nude mice were used to evaluate in vivo antitumor effects. We report that sorafenib and CuB exhibited synergistic effects on cellular proliferation inhibition and cell apoptosis induction, but not on cell cycle arrest. Furthermore, combination treatment enhanced levels of cleaved caspase 3 and cleaved caspase 9, but suppressed phosphorylation of STAT3. Epidermal growth factor, a potent stimulator of signal transducer and activator of transcription‐3 (STAT3), promoted cell viability and colony formation ability, whereas combination treatment exerted inhibitory effects on epidermal growth factor‐induced STAT3 phosphorylation. Finally, HepG2 xenograft mice cotreated with sorafenib and CuB exhibited reduced tumor progression without notable weight loss. In conclusion, sorafenib and CuB exert synergistic antitumor effects through a pathway that may involve STAT3 phosphorylation, and this may represent a promising therapeutic approach for treatment of HCC.

Diverse drug loading approaches for human heavy-chain ferritin (HFn), a promising drug nanocarrier, have been established. However, anti-tumor drug loading ratio and protein carrier recovery yield are bottlenecks for future clinical application. Mechanisms behind drug loading have not been elaborated. In this work, a thermally induced drug loading approach was introduced to load anti-tumor drug doxorubicin hydrochloride (DOX) into HFn, and 2 functionalized HFns, HFn-PAS-RGDK, and HFn-PAS. Optimal conditions were obtained through orthogonal tests. All 3 HFn-based proteins achieved high protein recovery yield and drug loading ratio. Size exclusion chromatography (SEC) and transmission electron microscopy (TEM) results showed the majority of DOX loaded protein (protein/DOX) remained its nanocage conformation. Computational analysis, molecular docking followed by molecular dynamic (MD) simulation, revealed mechanisms of DOX loading and formation of by-product by investigating non-covalent interactions between DOX with HFn subunit and possible binding modes of DOX and HFn after drug loading. In in vitro tests, DOX in protein/DOX entered tumor cell nucleus and inhibited tumor cell growth.