Functional importance of coacervation to convert calcium polyphosphate nanoparticles into the physiologically active state

Inorganic polyphosphates (polyP) are of increasing medical interest due to their unprecedented ability to exhibit both morphogenetic and ATP-delivering properties. However, these polymers are only physiologically active in the coacervate state, but not as amorphous nanoparticles (NP), the storage form of the polymer. Little is known about the mechanism of formation and interconversion of these two distinct polyP phases in the presence of metal ions. Based on in silico simulation studies, showing a differential clustering of polyP and calcium ions, the pH-dependent NP and coacervate formation of polyP was examined experimentally. Turbidimetric studies showed that Ca-polyP coacervate formation at pH 7 is a slow process compared to NP formation at pH 10. In FTIR spectra, the asymmetric stretching vibration signal of the internal (PO2)- units, which is present in the Ca-polyP coacervate formed at pH 7, disappears in the NP formed at pH 10 using the conventional method (dropping of a CaCl2 solution into a Na-polyP solution). Surprisingly, when reversing the procedure, adding Na-polyP to CaCl2, a coacervate is obtained at both pH 7 and pH 10, as confirmed by SEM and FTIR analyses. The (PO2)- signal also disappears when Ca-polyP-NP are exposed to peptides, leading to the transformation of the NP into the coacervate phase. From these results, a mechanistic model of pH-dependent coacervate and NP formation is proposed that considers not only electrostatic ion-ion but also ion-dipole interactions. Functional studies revealed a delayed polyP release kinetics for Ca-polyP-NP embedded in a hydrogel due to NP/coacervate conversion. Human A549 epithelial cells grown on the coacervate show increased proliferation and ATP production compared to cells cultured on particulate polyP. Ca-polyP NP taken up by endocytosis undergo intracellular coacervate transformation. Understanding the differential expression of the two polyP phases is of functional importance for the potential therapeutic application of this physiological, regeneratively active polymer.