How to avoid protein aggregation to improve cellular uptake of albumin-based conjugates: towards the rational design of cell-penetrable phosphorescent probes

Albumins are very promising carrier family for various types of small functional molecules, including (pro)drugs, sensors, and imaging agents. In this study, we demonstrate that one of the most crucial features of albumin—its ability to internalize into cancer cells—is strongly affected by its aggregation state. We used 14 different luminescent complexes (polynuclear heterometallic Ag(I)-Au(I) and Cu(I)-Au(I) clusters and a series of mononuclear water-soluble and hydrophobic Pt(II) complexes) able to form either covalent conjugates or non-covalent adducts with human serum albumin (HSA) to investigate the impact of preparation conditions on aggregation behavior of HSA. We demonstrated that preparation of non-aggregated HSA-based conjugates/adducts requires compliance with rather strict reaction conditions (aqueous solution with as low as possible organic co-solvent content and complex/HSA molar ratio not exceeding some critical value) while bypassing these rules (e.g., using the reaction mixtures enriched by organic solvent, high complex/HSA molar ratio or bulky hydrophobic molecules) may lead to HSA aggregation or even instability of the conjugates in solution. Further, we showed that non-aggregated HSA conjugates with water-soluble Pt complex easily internalize into cancer HeLa cells, while fully aggregated non-covalent HSA adducts with heterometallic Cu-Au clusters do not. Ultimately, in this study, we put forward two major points. First, we formulate general rules providing simple preparation route for non-aggregated cell-penetrable HSA-based conjugates, particularly, phosphorescent probes. Second, we demonstrate that the aggregation state of albumin is a very important parameter, which should be controlled while preparing its conjugates.