Redesigning human ferritin nanocages for therapeutic applications: from cancer treatment to hypercholesterolemia management

Protein-based nanoparticles are increasingly significant in nanomedicine due to their biocompatibility, specificity, and modifiability, offering innovative solutions to therapeutic challenges. Among these, human ferritin stands out for its versatility. Its hollow structure, along with the ability to modify both internal and external surfaces chemically or genetically, makes it an ideal candidate for a variety of applications, including targeted drug delivery, molecular imaging, immunotherapy, and vaccine development. Given these unique properties, this Ph.D. research explores the potential of human H ferritin (HFn) by modifying its surfaces for two distinct biomedical applications. The first objective focuses on enhancing ferritin’s ability to encapsulate hydrophobic drugs, addressing a major limitation in drug delivery systems. By incorporating four or six tryptophan residues per subunit, oriented towards the nanoparticle’s internal cavity, we increased its hydrophobicity, with the aim to improve its capacity to encapsulate hydrophobic chemotherapeutic agents. Detailed characterization revealed that only the variant with four tryptophan residues per subunit retained the ability to disassemble and reassemble correctly. To demonstrate the potential of this modification, we tested the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with anticancer properties. Our findings showed that this mutant was far more efficient at loading ellipticine compared to wild-type ferritin. We further evaluated the versatility of this nanoparticle by also encapsulating doxorubicin, a commonly used anticancer drug. Both ellipticine- and doxorubicin-loaded nanoparticles were tested on a promyelocytic leukemia cell line, where efficient drug uptake by the cells and the expected cytotoxic effects were observed. The second objective targets hypercholesterolemia, a major risk factor for cardiovascular disease, by focusing on Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), a protein that regulates cholesterol levels. PCSK9 decreases the number of LDL receptors on liver cells by promoting their degradation, impairing the clearance of LDL cholesterol from the bloodstream. To counteract this, HFn was genetically modified to display 24 copies of a 13-amino acid peptide (Pep2-8) on its surface, previously identified as the smallest PCSK9 inhibitor. Biochemical analysis confirmed precise control over nanoparticle size and morphology, as well as strong PCSK9-binding affinity in the high picomolar range. Functional studies in HepG2 liver cells showed enhanced LDL receptor recycling and LDL uptake, confirming the effectiveness of this multivalent nanoparticle in promoting cholesterol clearance. Together, these findings highlight the multifunctional nature of human H ferritin, making it as a promising platform that can be tailored for diverse therapeutic interventions, from targeted cancer treatment to the modulation of critical physiological pathways in various diseases. The ability to engineer ferritin nanoparticles to display different peptides or proteins further expands its versatility, opening avenues for the development of multifunctional therapeutics with the potential to target multiple pathways simultaneously.