Human Serum Albumin Protein Corona in Prussian Blue Nanoparticles

Prussian Blue nanoparticles (PBnps) are now popular in nanomedicine thanks to the FDA approval of PB. Despite the numerous papers suggesting or describing the in vivo use of PBnps, no studies have been carried out on the formation of a protein corona on the PBnp surface and its stabilizing role. In this paper, we studied qualitatively and quantitatively the corona formed by the most abundant protein of blood, human serum albumin (HSA). Cubic PBnps (41 nm side), prepared in citric acid solution at PB concentration 5 × 10^-4 M, readily form a protein corona by redissolving ultracentrifuged PBnp pellets in HSA solutions, with C_HSA ranging from 0.025 to 7.0 mg/mL. The basic decomposition of PBnp@HSA was studied in phosphate buffer at the physiological pH value of 7.4. Increased stability with respect to uncoated PBnps was observed at all concentrations, but a minimum C_HSA value of 3.0 mg/mL was determined to obtain stability identical to that observed at serum-like HSA concentrations (35-50 mg/mL). Using a modified Lowry protocol, the quantity of firmly bound HSA in the protein corona (hard corona) was determined for all the C_HSA used in the PBnp@HSA synthesis, finding increasing quantities with increasing C_HSA. In particular, an HSA/PBnp number in the 1500-2300 range was found for C_HSA 3.0-7.0 mg/mL, largely exceeding the 180 HSA/PBnp value calculated for an HSA monolayer on a PBnp. Finally, the stabilization brought by the HSA corona allowed us to carry out pH-spectrophotometric titrations on PBnp@HSA in the 3.5-9-0 pH range, revealing a pKa value of 6.68 for the water molecules bound to the Fe³⁺ centers on the PBnp surface, whose deprotonation is responsible for the blue-shift of the PBnp band from 706 nm (acidic solution) to 685 nm (basic solution).