<i>In Vivo</i> Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures

Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used <i>in vitro</i> for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their <i>in vivo</i> stability, circulation, and tissue distribution. In an effort to evaluate CSANs <i>in vivo</i>, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their <i>in vivo</i> tissue localization by microPET/CT imaging, and determined their <i>ex vivo</i> organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR²) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. <i>In vitro</i> binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while <i>in vivo</i> microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA­[⁶⁴Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN’s binding affinity toward U-87 MG glioblastoma cells <i>in vitro</i>. A significant time dependent tumor accumulation of ⁶⁴Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an <i>in vivo</i> profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for <i>in vivo</i> drug delivery and imaging applications.