Figure 3.

Synthesis of antibody targeted nano contrast agents.

Notes: Steps 1 and 2: After NHS/DCC activation of PMLA’s pendant carboxylates, Gd-DOTA-PEG600-amine and/or 2-mercapto-1-ethylamine (MEA) were attached through amide linkage to form Preconjugate-1 and Preconjugate-2. Steps 3–5: Mal-functionalized mAbs (Cetuximab and MsTfR) were attached to Preconjugate-1 and Preconjugate-2 in phosphate buffer (100 mM sodium phosphate, pH 6.3) for 30 minutes at ambient temperature. After attaching antibodies, pH was adjusted to 5.5 and intermediates mal-PEG600(Gd-DOTA)₃ and mal-PEG10000(Gd-DOTA)₇ were conjugated followed by Rhodamine-mal via thioether bond at 4°C overnight. Finally, excess thiol groups were masked using PDP and the final product was purified by PD-10 columns in PBS (pH 7.4). Cetuximab was chosen for EGFR targeting and anti-mouse TfR mAb for specific binding to mouse TfR for transcytosis across vascular endothelial layer (BBB crossing).

Abbreviations: PMLA, Polymalic acid; mal, maleimide; PDP, 2-pyridyldithiopropionate; TfR, Transferrin receptor; BBB, blood–brain barrier.