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.

中文翻译：

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铁蛋白纳米笼与生物正交共轭的血管靶向和成像。

将生物学上正交的官能团遗传掺入大分子中具有潜在地产生亲和配体，造影剂或治疗药物的有效，可控制，可再现，位点特异性缀合的潜力。在这里，我们将这种方法应用于铁蛋白，铁蛋白是一种普遍存在的铁存储蛋白，可自我组装成具有出色稳定性，尺寸均一性（12 nm）以及可装载和运输各种无机和有机货物的内生能力的多聚体纳米笼。非天然氨基酸4-叠氮基苯丙氨酸（4-AzF）被掺入人铁蛋白轻链（hFTL）的不同位点，以允许含炔的小分子或亲和配体与纳米笼的外表面特异性结合。通过筛选变体文库中无铜咔嗒共轭效率的方法，评估了4-AzF残基的最佳位置。发现一种工程铁蛋白hFTL-5X可容纳每个纳米笼子约14个小分子荧光团（AlexaFluor 488）和3-4个IgG分子。小鼠血管内注射放射性标记的hFTL-5X携带抗细胞粘附分子ICAM-1的抗体，但不能控制IgG，由于ICAM-1的高基础表达而使其能够特异性靶向肺（43.3±6.99 vs 3.48±0.14％ID / g（相对于IgG）。用已知刺激炎症性ICAM-1过表达的内毒素治疗小鼠，可使肺部靶向性提高2倍（84.4±12.89 vs 43.3±6.99％ID / g）。同样地，注入荧光粉