Calcium orthophosphates, such as hydroxyapatite (HAP), have been shown to follow nonclassical nucleation and crystallization mechanisms involving intermediate species, including solute prenucleation species (PNS) that precede solid transient phases. Characterizing these transient soluble and solid phases in situ remains a challenge due to their short-lived nature. In this study, we combine 43Ca isotopic enrichment with dynamic nuclear polarization (MAS-DNP) under cryogenic conditions to enhance 43Ca sensitivity and capture transient calcium species in their native state in the course of HAP formation. Fine calibration of the Hartmann–Hahn cross-polarization conditions (low vs high radio frequency powers) enables us to distinguish different calcium-based phases based on the strength of their 43Ca quadrupolar couplings. Hence, we can record the 43Ca NMR fingerprint of submillimolar concentrations of PNS and two-dimensional 1H–43Ca correlation NMR spectra of calcium phosphate solid phases can be recorded within hours. Thanks to this 2D analysis, the first nucleating solid phase is identified as an amorphous calcium phosphate (ACP) rich in acidic orthophosphate ions and water molecules. Moreover, the core-layer organization of the final HAP phase is also evidenced allowing to distinguish the 43Ca spectral fingerprints of the crystalline hydroxyapatite core from the amorphous hydrated layer. This work demonstrates the potential of 43Ca MAS-DNP for characterizing the complex nucleation and growth mechanisms of calcium-containing materials, thus, offering new opportunities for studying similar systems in environmental and biological contexts.

Biomimetic silica-peptide nanocomposites are promising materials for applications in drug delivery and enzyme encapsulation due to their biocompatibility, tunable morphologies, and unique structural characteristics. However, the structural dynamics of the peptide scaffold remain largely elusive, impeding rational biomimetic materials design. This shortcoming is not the least due to a lack of methods that can access such heterogeneous systems with dynamics on a wide range of time scales. Among the most studied candidates are silica particles templated by the diatom-derived peptide R5, known for its ability to guide silica precipitation under mild, toxicologically friendly conditions, leading to silica capsules filled with a peptide scaffold. Here, we describe the structural dynamics of R5 within its self-assemblies and the silica particles it templates with a combination of advanced magnetic resonance methods, including 13C-direct detected NMR, site-directive spin-labeling EPR, and sensitivity-enhanced solid-state NMR. We provide evidence that R5 self-assemblies form condensed phases with liquid-like dynamics both before and after silica encapsulation. Our suite of methods allowed us to access R5/silica composites over a comprehensive range of time scales. These results demonstrate that R5 retains a remarkable degree of internal dynamics, with distinct regions of solid-like and liquid-like behavior even within the silica particles. Specifically, the peptide scaffold comprises three dynamic species: (i) solid-like at the peptide-silica interface, (ii) liquid-like mobility within the scaffold core, and (iii) intermediate dynamics at the boundary regions between core and interface species. Our findings rationalize the high mobility of guest molecules, such as drugs or enzyme substrates, within R5-silica nanoparticles, which is crucial for their functionality in controlled release and catalytic applications. This understanding paves the way for improved rational design considerations for advanced nanomaterials and expands our knowledge of biomimetic mineralization mechanisms. At the same time, the methodological approach can be useful for many types of peptide-guided biominerals, bridging fundamental biochemistry with biotechnological innovation.

Calcium ion complexation in aqueous solutions is of paramount importance in biology as it is related to cell signaling, muscle contraction, or biomineralization. However, Ca2+-complexes are dynamic soluble entities challenging to describe at the molecular level. Nuclear magnetic resonance appears as a method of choice to probe Ca2+-complexes. However, 43Ca NMR exhibits severe limitations arising from the low natural abundance coupled to the low gyromagnetic ratio and the quadrupolar nature of 43Ca, which overall make it a very unreceptive nucleus. Here, we show that 43Ca dynamic nuclear polarization (DNP) NMR of 43Ca-labeled frozen solutions is an efficient approach to enhance the NMR receptivity of 43Ca and to obtain structural insights about calcium ions complexed with representative ligands including water molecules, ethylenediaminetetraacetic acid (EDTA), and l-aspartic acid (l-Asp). In these conditions and in combination with numerical simulations and calculations, we show that 43Ca nuclei belonging to Ca2+ complexed to the investigated ligands exhibit rather low quadrupolar couplings (with CQ typically ranging from 0.6 to 1 MHz) due to high symmetrical environments and potential residual dynamics in vitrified solutions at a temperature of 100 K. As a consequence, when 1H→43Ca cross-polarization (CP) is used to observe 43Ca central transition, “high-power” νRF(43Ca) conditions, typically used to detect spin 1/2 nuclei, provide ∼120 times larger sensitivity than “low-power” conditions usually employed for detection of quadrupolar nuclei. These “high-power” CPMAS conditions allow two-dimensional (2D) 1H–43Ca HetCor spectra to be readily recorded, highlighting various Ca2+–ligand interactions in solution. This significant increase in 43Ca NMR sensitivity results from the combination of distinct advantages: (i) an efficient 1H-mediated polarization transfer from DNP, resembling the case of low-natural-abundance spin 1/2 nuclei, (ii) a reduced dynamics, allowing the use of CP as a sensitivity enhancement technique, and (iii) the presence of a relatively highly symmetrical Ca environment, which, combined to residual dynamics, leads to the averaging of the quadrupolar interaction and hence to efficient high-power CP conditions. Interestingly, these results indicate that the use of high-power CP conditions is an effective way of selecting symmetrical and/or dynamic 43Ca environments of calcium-containing frozen solution, capable of filtering out more rigid and/or anisotropic 43Ca sites characterized by larger quadrupolar constants. This approach could open the way to the atomic-level investigation of calcium environments in more complex, heterogeneous frozen solutions, such as those encountered at the early stages of calcium phosphate or calcium carbonate biomineralization events.

In this study, nuclear magnetic resonance (NMR) is used to investigate the crystallisation behaviour of aspirin within a mesoporous SBA-15 silica material. The potential of dynamic nuclear polarisation (DNP) experiments is also investigated using specifically designed porous materials that incorporate polarising agents within their walls. The formation of the metastable crystalline form II is observed when crystallisation occurs within the pores of the mesoporous structure. Conversely, bulk crystallisation yields the most stable form, namely form I, of aspirin. Remarkably, the metastable form II remains trapped within the pores of mesoporous SBA-15 silica material even 30 days after impregnation, underscoring its persistent stability within this confined environment.