Branched Polymeric Prenucleation Assemblies Initiate Calcium Phosphate Precipitation

The formation of crystalline calcium phosphate (CaP) has recently gained ample attention as it does not follow the classic nucleation-and-growth mechanism of solid formation.¹ Instead, the precipitation mechanisms can involve numerous intermediates, including soluble prenucleation species such as clusters, oligomers or dense liquid phases in presence of organic additives. However, structural characteristics, stability, and transformation of such solution-state precursors remain largely undisclosed. Recently our group reported that the proportion and lifetime of CaP prenucleation clusters is determined by simple synthesis parameters, namely ionic concentrations, pH and ionic strength.² Moreover, our consortium has shown the strength of NMR approaches to provide structural and dynamical information at the molecular scale. As an example, we have shown through <i>in situ</i> ³¹P NMR that simulated body fluid (SBF) is subject to the formation and aggregation of PNC for 24h after its preparation³ and that dissolution dynamic nuclear polarization (dDNP) combined with ³¹P NMR is a suitable technique to monitor fast precipitation processes (&lt; 20 s) and to access to the size and dynamical parameters of PNC, such as formation, aggregation and exchange rates.⁴<br/>In this communication, we report a detailed and comprehensive characterization of the sequential events involved in calcium phosphate crystallization starting from the very early prenucleation stage. We integrated an extensive set of time-resolved methods, including NMR, SAXS, DLS, cryo-TEM, and calcium-potentiometry to show that CaP nucleation is initiated by the transformation of “branched” polymeric prenucleation assemblies into amorphous calcium phosphate spheres. Such a mineralization process starts with the spontaneous formation of so-called nanometric prenucleation clusters (PNC) that later assemble into those soluble branched polymeric assemblies without calcium ion uptake from the solution. Importantly, these macromolecular species are invisible to many techniques (NMR, turbidity, calcium-potentiometry) but can readily be evidenced by time-resolved SAXS and cryo-TEM. We find that the branched polymeric assemblies constitute the origin of amorphous calcium phosphate (ACP) precipitation through an unexpected process: spontaneous dissolution is followed by local densification of 100-200 nm wide domains leading to ACP spheres of similar size. Hence, our study demonstrates that ACP nucleation does not necessarily proceed through simple aggregation of prenucleation clusters. Finally, we show that the timing of the successive events involved in the CaP mineralization pathway can be kinetically controlled by the Ca²⁺/Pi molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.<br/> <br/>REFERENCES<br/>¹ Habraken, W. J. <i>et al</i>. <i>Nature Commun.</i> 2013, <i>4</i>(1), 1507.<br/>² Georges, T. <i>et al.</i> <i>Cryst. Growth Des. </i>2024, <i>24</i>(9), 3865-3875.<br/>³ Epasto, L. M., Georges, T. <i>et al.</i> <i>Anal. Chem. </i>2021, <i>93</i>(29), 10204-10211<br/>⁴ Weber, E. M. et al. <i>Anal. Chem. </i>2020, <i>92</i>(11), 7666-7673