Computational chemistry of phospholipid mineralization

Abstract: Previously, we have found that cell membrane nanofragments (phospholipids) were the nucleation site for bone formation (Hara ES et al., ACS Biomater Sci Eng, 2018). In addition, we showed that cell membrane nanofragments could promote bone-like mineral (calcium phosphate) formation in only 1 day (Hara ES et al., J Mater Chem B, 2018). However, the mineralization mechanisms of phospholipids are still unclear. This study aimed to evaluate the ability of different phospholipids to mineralize in vitro, and to analyze the energy required for phospholipid cleavage and calcium phosphate formation using in silico quantum chemistry-based tools. Chloroform-dissolved phospholipids were applied onto a glass-bottom dish, and after complete dry, a 2 mM CaCl2 solution was poured into the dish, which was incubated in at 37C, 5% CO2 and 98% humidity for 2 days. The phospholipid/mineral complexes were then collected in small tubes and centrifugally-washed 3 times with ultra-pure water (Milli-Q) and dissolved in 0.1% HCl for quantitative analysis of calcium levels using a simultaneous multi-element analysis atomic absorption spectrophotometer (AAS). A detailed analysis of the cleavage reaction kinetics focusing on the steric factor of each phospholipid molecule was performed to evaluate the affinity of each molecule for binding to calcium ions. The Gaussian16 molecular orbital program was used at the B3LYP/6-31(d,p) level of theory. The results showed that among several phospholipids, phospholipid X showed the highest mineralization ability in vitro, as determined by AAS. Quantum chemical calculations revealed that the mineralization reaction of phospholipid X probably proceeded in the following order: (1) binding of Ca ions (2) removal of the hydrophilic head, (3) removal of the hydrophobic tail, and formation of calcium phosphates. These results reveal important steps of phospholipid mineralization. Future studies are required to clarify the exact mechanisms of phospholipid mineralization.