Engineered Human Heavy-Chain Ferritin with Half-Life Extension and Tumor Targeting by PAS and RGDK Peptide Functionalization

Abstract

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.

Mechanism Study of Thermally Induced Anti-Tumor Drug Loading to Engineered Human Heavy-Chain Ferritin Nanocages Aided by Computational Analysis

“…Two functionalized HFns were more prone to aggregation in the heating process, suggesting slightly decreased thermal stabilities. This could be ascribed to the 'flip to flop' phenomenon in functionalized HFns, where E-helix with inserted functional peptide are extruded outside HFn nanocage, as was discovered in our previous work [20]. Hydrophobic interactions of 4 helices E around each hydrophobic channel in natural 'flip' HFn have been proven to contribute to HFn stability [29,30].…”

“…Three HFn-based proteins, HFn, HFn-PAS, and HFn-PAS-RGDK were designed as in a previous work [20]. Escherichia coli (E. coli) BL21 (DE3) (Tiangen Biotech, Beijing, China) was the expression host.…”

“…MDA-MB-231 cells in exponential growth phase were utilized in intracellular distribution analysis and cytotoxicity assay. Procedures of these two assays were the same as in a previous work using another tumor cell line [20]. A well and cell viability were calculated using the following equations.…”

Supramolecular Systems for Gene and Drug Delivery

“…Several biomaterial-based supramolecular systems (cyclodextrins [ 1 ], calixarenes [ 2 , 3 ], polymers [ 4 ], carbon nanotubes [ 5 ], nanoparticles [ 6 , 7 ], liposomes [ 3 , 8 ], nanogels [ 9 ], and nanocomplexes [ 10 ], among others) have been widely used for biomedical applications, such as gene and drug delivery. Numerous researchers have developed novel supramolecular systems for enhancing their biocompatibility and pharmacological activity, thus increasing their therapeutic properties.…”

“…S. Yin et al [ 7 ] prepared NPs based on the insertion of two types of functional peptides, half-life extension peptide PAS and tumor-targeting peptide RGDK (Arg-Gly-Asp-Lys), into human heavy-chain ferritin (HFn) at the C-terminal through flexible linkers with two distinct enzyme-cleavable sites. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase in growth inhibition.…”