Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging

• Published in: Bioconjugate chemistry 2018-04, Vol.29 (4), p.1209-1218
• Main Authors: Khoshnejad, Makan, Greineder, Colin F, Pulsipher, Katherine W, Villa, Carlos H, Altun, Burcin, Pan, Daniel C, Tsourkas, Andrew, Dmochowski, Ivan J, Muzykantov, Vladimir R
• Format: Article
• Language: English
• Subjects: Affinity; Alkynes; Alkynes - chemical synthesis; Alkynes - chemistry; Amino acids; Animals; Azides - chemical synthesis; Azides - chemistry; Cell adhesion; Cell adhesion molecules; Chemical compounds; Click Chemistry - methods; Conjugation; Contrast agents; Endotoxins; Ferritin; Ferritins - chemical synthesis; Ferritins - chemistry; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorophores; Functional groups; Gene expression; Humans; Immunoconjugates - chemistry; Immunoglobulin G; Immunoglobulins; Inflammation; Inflammation - diagnostic imaging; Injection; Intercellular adhesion molecule 1; Intercellular Adhesion Molecule-1 - analysis; Ligands; Lung - diagnostic imaging; Lungs; Macromolecules; Medical imaging; Mice; Molecular chains; Nanostructures - chemistry; Optical Imaging - methods; Pharmacology; Phenylalanine - analogs & derivatives; Phenylalanine - chemical synthesis; Phenylalanine - chemistry; Proteins
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.8b00004

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.