High affinity zoledronate-based metal complex nanocrystals to potentially treat osteolytic metastases

Formation of several materials, denoted as bisphosphonate-based coordination complexes (BPCCs), resulted from the reaction between clinically employed bisphosphonate, zoledronate (ZOLE) and bioactive metals (M²⁺ = Ca²⁺, Mg²⁺ and Zn²⁺). Six ZOLE-based BPCCs were synthesized using different variables (M²⁺ : ZOLE molar ratio, temperature, pH, and anion) and their structures were elucidated by single crystal X-ray diffraction (ZOLE-Ca forms I and II, ZOLE-Mg forms I and II, and ZOLE-Zn forms I and II). The dissolution of the ZOLE-based BPCCs was compared to that of ZOLE (Reclast®). Most of the ZOLE-based BPCCs (60-85%, in 18-24 h) present a lower dissolution and equilibrium solubility than ZOLE (∼100%, 30 min) in phosphate buffered saline (PBS), while a significantly higher dissolution is observed in acidic media (88% in 1 h). This suggests the ability to release the ZOLE content in a pH-dependent manner. Moreover, a phase inversion temperature (PIT)-nano-emulsion synthesis was performed, which demonstrated the ability to significantly decrease the crystal size of ZOLE-Ca form II from a micron-range (∼200 μm) to a nano-range (∼150 d nm), resulting in nano-Ca@ZOLE. Furthermore, low aggregation of nano-Ca@ZOLE in 10% fetal bovine serum (FBS) : PBS after 0, 24 and 48 h was demonstrated. Additionally, nano-Ca@ZOLE showed an ∼2.5x more binding to hydroxyapatite (HA, 36%) than ZOLE (15%) in 1 d. The cytotoxicity of nano-Ca@ZOLE against MDA-MB-231 (cancer cell model) and hFOB 1.19 (normal osteoblast-like cell model) cell lines was investigated. The results demonstrated significant cell growth inhibition for nano-Ca@ZOLE against MDA-MB-231, specifically at a low concentration of 3.8 μM (%RCL = 55 ± 1%, 72 h). Under the same conditions, the nanocrystals did not present cytotoxicity against hFOB 1.19 (%RCL = 100 ± 2%). These results evidence that nano-ZOLE-based BPCCs possess viable properties in terms of structure, dissolution, stability, binding, and cytotoxicity, which render them suitable for osteolytic metastasis therapy.