Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging

Abstract

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae

“…Therefore, both Ab/(Ft/SOD) and (Ab + SOD)/Ft displayed similar size characteristics of 20–25 nm in diameter with PDI 0.20–0.24. Earlier we have demonstrated that on average three molecules of IgG can be conjugated to the surface of Ft particle under the described conditions [40]. Therefore, one Ab/(Ft/SOD) particle contains on average 3 and 2 molecules of IgG and SOD, respectively.…”

Ferritin as a Platform for Creating Antiviral Mosaic Nanocages: Prospects for Treating COVID‐19

“…Consequently, interest in application of ferritin nanocage in drug delivery and MRI imaging has been increasing. [12,[37][38][39][40][41][42] Yet, its use for making antiviral mosaic nanocages to target viral particles has not been demonstrated. I will discuss four strategies to engineer ferritin and create antiviral mosaic nanocages as therapeutic candidates with the aim to address the current global pandemic of SARS-CoV-2.…”

“…[48] On the other hand, protein nanocages and ferritins furnished with multiple copy of a single antibody have been described for targeting specific cells and drug delivery. [39,40,49,50] However, mosaic nanocages to deliver a cocktail of antibodies and treat viral infections have yet to be described. These nanocages could significantly improve the efficacy of antibody therapy by preventing the emergence of scape variants and the ADE of infection.…”

“…This strategy has been used to add monoclonal antibodies to the N terminus of human L-chain ferritin for vascular targeting and imaging. [39] Alternatively, if a uAA like p-acetylphenylalanine (pAcF) is added to the N terminus of ferritin subunits and the C terminus of antibodies or their Fabs, chemical linkers could be used to furnish the surface of nanocages with antibodies using the method described by Kim et al [55] (Figure 3d). The neutralizing antibodies that could be used to furnish ferritin nanocage and create therapeutics for SARS-CoV-2 include OV2-2130, COV2-2165, COV2-2196, and CR3022.…”

Ferritin: A Platform for MRI Contrast Agents Delivery

“…To increase targeted ferritin delivery, more selective ligands of cell surface markers, such as peptides and antibodies, have been conjugated to the external surface of ferritin [25]. In this contest, Khoshnejad M. et al have shown high pulmonary endothelial targeting in vivo, with no noticeable undesired effects, by functionalizing ferritin with antibodies specific for ICAM-1 [26]. The present review aims to highlight recent advances achieved using both H-and L-ferritins, loaded with paramagnetic metals or complexes, as highly sensitive MRI probes ( Figure 1).…”