Long circulating Ferritin Nanocage with 'Protein cloud'

Abstract

Protein nanocages have a wide range of applicability in research due to their favourable features, such as high biocompatibility, low toxicity profiles and capacity for genetic and chemical modification. Such an example are ferritin nanocages, endogenous in nature, that have been modified for use as delivery vehicles of therapeutic drugs and peptides in research. Here we have developed ferritin nanocages with an extended pharmacokinetic profile that can be used as a platform for application in research.

Protein-based agents are prone to rapid degradation in vivo and approaches such as PEGylation that can overcome this issue are continuously being pursued. Due to the heterogeneous nature of PEG itself, chemical conjugation of PEG leads to poor quality control of its products. However, fusion of XTEN, a peptide composed of hydrophilic amino acids, can be done in a one step recombinant protein synthesis with high yields using bacteria E. coli. Using previously developed XTEN peptides, a protein corona covering the outer surface of the nanocage was formed. Ferritin nanocages with the protein corona were synthesized through bacterial expression of the hybrid gene consisting of human ferritin heavy chain (hFTH), linker (glycine- rich peptide) and XTEN genes. The self-assembly of of hfTH leads to the formation of nanocages (12 nm in diameter), and varying lengths of XTEN peptides attached at the C-termini constitutes an outer layer of protein corona, resulting in the construction of FTH-XTEN nanocages.

These modified ferritin nanocages show lengthened half-lives in vivo and overall improved pharmacokinetic profiles as compared to ferritin alone. The half-lives of nanocages increase in correlation to increasing length of XTEN peptide attached. In vitro and in vivo characterizations of the developed FTH-XTEN nanocages are reported here, and these nanocages show potential for various applications in delivery of drugs and therapeutic peptides.