A Versatile Platform to Generate Prodrugs with Rapid and Precise Albumin Hitchhiking and High Cargo Loading for Tumor-Targeted Chemotherapy.

Due to its tumor homing and long serum half-life, albumin is an ideal drug carrier for chemotherapy. For endogenous albumin hitchhiking with high cargo loading, a trimeric albumin-binding domain (ABD), i.e., ABD-Tri is designed by fusing an ABD with high specificity and affinity for albumin to a self-trimerizing domain (Tri) with an additional cysteine residue. ABD-Tri is highly (40 mg L -1 ) expressed as soluble and trimeric proteins in Escherichia coli (E. coli). Once mixed together, ABD-Tri rapidly and specifically forms a stable complex with albumin under physiological conditions without obviously changing its receptor- and cell-binding and tumor-homing properties. Maleimide-modified prodrugs are highly effectively conjugated to ABD-Tri to produce homogenous ABD-Tri-prodrugs with triple cargo loading under physiological conditions by thiol-maleimide click chemistry. Unlike the maleimide moiety, which can only mediate time- and concentration-dependent albumin binding, ABD-Tri mediated fast (within several minutes) albumin binding of drugs even at extremely low concentrations (µg mL -1 ). Compared to maleimide-modified prodrugs, ABD-Tri-prodrugs exhibit better tumor homing and greater in vivo antitumor effect, indicating that conjugation of chemical drug to ABD-Tri outperforms maleimide modification for endogenous albumin hitchhiking. The results demonstrate that ABD-Tri may serve as a novel platform to produce albumin-binding prodrugs with high cargo-loading capacity for tumor-targeted chemotherapy.