1 research outputs found
Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging
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