Hydroxycitrate is a Unique Inhibitor of Pathologically-Relevant Brushite Crystallization

The unique interactions between (macro)molecules and the surfaces of crystalline materials is a common mechanism for controlling the formation of natural, biological, and synthetic crystals. These species, generally referred to as modifiers, can either inhibit or promote crystallization in ways that achieve properties unattainable by alternative processes, or alter nucleation to produce metastable polymorphs or solvates. In the field of pathological crystallization, modifiers can be native species or rationally designed molecules for the therapeutic purpose of suppressing crystal nucleation and/or growth. Here, we assess the effects of hydroxycitrate as a modifier of brushite (calcium phosphate dihydrate) crystallization and demonstrate its superior efficacy as an inhibitor compared to citrate, its molecular analogue present in urine naturally and commonly administered as a preventative therapy for calcium-based kidney stones. Using a combination of bulk crystallization, microfluidics, and atomic force microscopy, we show that hydroxycitrate is capable of completely suppressing brushite nucleation and operates as an effective growth inhibitor at concentrations nearly one order of magnitude lower than citrate. Our findings reveal a concentration-dependent mode of action for hydroxycitrate which at low supersaturation induces the formation of defects (i.e., surface voids) far from nucleation centers that are not observed at high supersaturation. Kinetic studies reveal that hydroxycitrate fully suppresses two of the three principal growth directions, whereas citrate only suppresses one direction. For crystallographic growth directions where modifiers are moderately effective, both citrate and hydroxycitrate inhibit crystallization by as much as 60%, leading to markedly reduced rates of growth and concomitant changes in crystal habit. Collectively, the results of this study open avenues into the design of potent inhibitors of pathological crystallization with potential implications for other biominerals.