Structural characterization of amorphous calcium phosphate-based calciprotein particles stabilized by proteins

• Biomimetic calciprotein particles (CPPs) were synthesized with fetuin-A and albumin. • Magnesium ions dramatically extend the lifetime of CPP1. • CPPs form a 50 nm "raspberry-like" structure with a disordered, lacking a core-shell. • Fetuin-A preferentially incorporates into CPPs over albumin. • Fetuin-A and albumin stabilize ACP, control CPP size, and influence particle structure. Hybrid nanoparticles made of amorphous calcium phosphate (ACP) and proteins, known as calciprotein particles (CPPs), serve as a valuable resource in the body to prevent the formation of unwanted calcifications in our blood. The nanoscale architecture of these endogenous colloidal particles is influenced by the presence of proteins such as fetuin-A and albumin in the synthetic medium, which stabilize ACP in terms of both size and crystallinity. However, a detailed understanding of the structural role of these proteins within the inorganic ACP component is lacking in the literature, despite its potential to provide important insights into the formation mechanism and stability of such hybrid nanoparticles. This work unravels the architecture at the nanoscale of biomimetic CPPs prepared upon formation of ACP in the presence of bio-relevant concentrations of fetuin-A and albumin, while keeping constant the concentration of the former and varying the latter. A multi-technique approach was employed to gain a comprehensive understanding of the system, investigating the evolution of CPPs in solution over time and their structural characteristics by utilizing several techniques, including cryo-electron microscopy and small-angle neutron scattering experiments, among others. The results show that we managed to obtain bio-mimetic CPPs made of ACP and proteins, which have an extended stability in solution in terms of size and crystallinity. Fetuin-A and albumin do not form a homogeneous layer on the outside of the inorganic core but generate a raspberry-like structure in which the ACP clusters stabilized by the proteins self-assemble into nanoparticles.