High-Affinity Extended Bisphosphonate-Based Coordination Polymers as Promising Candidates for Bone-Targeted Drug Delivery.

Extended bisphosphonate-based coordination polymers (BPCPs) were produced when 1,1'-biphenyl-4,4'-bisphosphonic acid (BPBPA), the analogue of 1,1'-biphenyl-4,4'-dicarboxylic acid (BPDC), reacted with bioactive metals (Ca2+, Zn2+, and Mg2+). BPBPA-Ca (11 Å × 12 Å), BPBPA-Zn (10 Å × 13 Å), and BPBPA-Mg (8 Å × 11 Å) possess channels that allow the encapsulation of letrozole (LET), an antineoplastic drug that combined with BPs treats breast-cancer-induced osteolytic metastases (OM). Dissolution curves obtained in phosphate-buffered saline (PBS) and fasted-state simulated gastric fluid (FaSSGF) demonstrate the pH-dependent degradation of BPCPs. Specifically, the results show that the structure of BPBPA-Ca is preserved in PBS (∼10% release of BPBPA) and collapses in FaSSGF. Moreover, the phase inversion temperature nanoemulsion method yielded nano-Ca@BPBPA (∼160 d. nm), a material with measurably higher (>1.5x) binding to hydroxyapatite than commercial BPs. Furthermore, it was found that the amounts of LET encapsulated and released (∼20 wt %) from BPBPA-Ca and nano-Ca@BPBPA are comparable to those of BPDC-based CPs [i.e., UiO-67-(NH2)2, BPDC-Zr, and bio-MOF-1], where other antineoplastic drugs have been loaded and released under similar conditions. Cell viability assays show that, at 12.5 µM, the drug-loaded nano-Ca@BPBPA exhibits higher cytotoxicity against breast cancer cells MCF-7 and MDA-MB-231 [relative cell viability (%RCV) = 20 ± 1 and 45 ± 4%] compared with LET (%RCV = 70 ± 1 and 99 ± 1%). At this concentration, no significant cytotoxicity was found for the hFOB 1.19 cells treated with drug-loaded nano-Ca@BPBPA and LET (%RCV = 100 ± 1%). Collectively, these results demonstrate the potential of nano-Ca@BPCPs as promising drug-delivery systems to treat OM or other bone-related diseases because these present measurably higher affinity, allowing bone-targeted drug delivery under acidic environments and effecting cytotoxicity on estrogen receptor-positive and triple-negative breast cancer cell lines known to induce bone metastases, without significantly affecting normal osteoblasts at the metastatic site.

Design of Extended Bisphosphonate-Based Coordination Polymers as Bone-Targeted Drug Delivery Systems for Breast Cancer-Induced Osteolytic Metastasis and Other Bone Therapies.

The coordination between benzene 1,4-bis(bisphosphonic acid) (BBPA), the bisphosphonate (BP) analogue of benzene 1,4-dicarboxylic acid (BDC), and bioactive metals led to the formation of extended bisphosphonate-based coordination polymers (BPCPs). Four distinct crystalline phases were obtained, namely, BBPA-Ca forms I and II, BBPA-Zn, and BBPA-Mg. Among these, BBPA-Ca forms I (7 × 9 Å2) and II (8 × 12 Å2) possess channels large enough to encapsulate 5-fluorouracil (5-FU), a drug prescribed in combination with BPs to treat breast cancer-induced osteolytic metastases (OM). Dissolution curves show a 14% release of BBPA from BBPA-Ca form II in phosphate-buffered saline, while ∼90% was released in fasted-state simulated gastric fluid. These results suggest that this material is relatively stable in neutral environments yet collapses in acidic conditions. Moreover, the phase inversion temperature method decreased the particle size of BBPA-Ca form II, resulting in nano-Ca@BBPA (∼134 d.nm). Binding assays showed a higher affinity of nano-Ca@BBPA (∼97%) to hydroxyapatite than BBPA (∼70%) and significantly higher binding than commercial BPs, zolendronic (3.0×), and risedronic (2.4×) acids after 24 h. Furthermore, both BBPA-Ca form II and nano-Ca@BBPA presented comparable drug loading and release (∼30 wt % 5-FU) relative to BDC-based CCs (UiO-66, MIL-53, and BDC-Zr) where other pharmaceutical compounds (caffeine, ibuprofen, aspirin, and α-cyano-4-hydroxycinnamic acid) have been encapsulated. Cell viability assays established that drug-loaded nano-Ca@BBPA increases the cytotoxicity of a triple-negative human breast cancer cell line (MDA-MB-231) when compared to 5-FU (%RCV = 8 ± 5 vs 75 ± 1% at a 100 µM). At the same concentration, no significant decrease in cell viability was observed for normal human osteoblast-like hFOB 1.19 cells (%RCV = 85 ± 1%). Collectively, these results demonstrate the feasibility of nano-Ca@BBPA as a potential drug delivery system (DDS), with high affinity to bone tissue, to treat bone-related diseases such as OM.

Polymorphic control in titanium dioxide particles.

The hydrolysis-condensation reaction of TiO2 was adapted to the phase inversion temperature (PIT)-nano-emulsion method as a low energy approach to gain control over the size and phase purity of the resulting metal oxide particles. Three different PIT-nano-emulsion syntheses were designed, each one intended to isolate high purity rutile, anatase, and brookite phase particles. Three different emulsion systems were prepared, with a pH of either strongly acidic (H2O : HNO3, pH ∼0.5), moderately acidic (H2O : isopropanol, pH ∼4.5), or alkaline (H2O : NaOH, pH ∼12). PIT-nano-emulsion syntheses of the amorphous TiO2 particles were conducted under these conditions, resulting in average particle diameter distributions of ∼140 d nm (strongly acidic), ∼60 d nm (moderately acidic), and ∼460 d nm (alkaline). Different thermal treatments were performed on the amorphous particles obtained from the PIT-nano-emulsion syntheses. Raman spectroscopy and powder X-ray diffraction (PXRD) were employed to corroborate that the thermally treated particles under H2O : HNO3 (at 850 °C), H2O : NaOH (at 400 °C), and H2O : isopropanol (at 200 °C) yielded highly-pure rutile, anatase, and brookite phases, respectively. Herein, an experimental approach based on the PIT-nano-emulsion method is demonstrated to synthesize phase-controlled TiO2 particles with high purity employing fewer toxic compounds, reducing the quantity of starting materials, and with a minimum energy input, particularly for the almost elusive brookite phase.

Functionalization of Titanium Dioxide by In Situ Surface Crystallization of Bisphosphonate-Based Coordination Complexes.

Functionalization of highly pure rutile phase titanium dioxide (TiO2) particles with a selected bisphosphonate-based coordination complex (BPCC), ZOLE-Ca form II, was achieved through in situ surface crystallization. The hydrothermal reaction of the selected BPCC was carried out in the presence of photoactivated rutile phase TiO2 by ultraviolet irradiation. The reaction time was varied to control the crystal growth of the BPCC around the TiO2 core, resulting in a functionalized material with different shell thicknesses: TiO2-core:nano-Ca@ZOLE-shell-† (5 min) and TiO2-core:nano-Ca@ZOLE-shell-‡ (10 min). The crystal phase assessment of the BPCC and the polymorphic phase purity of the metal oxide were determined after immobilization through Raman spectroscopy and powder X-ray diffraction. The results initially suggested that the crystallization of a shell comprising the selected BPCC surrounding a highly pure rutile phase TiO2 core was achieved through controlled in situ surface crystallization. Morphological changes, elemental composition and exact atomic distribution in the functionalized materials were addressed employing scanning electron microscopy coupled with energy-dispersive spectroscopy. These analyses unambiguously confirmed that after 5 min, successful incorporation of a thin BPCC shell on the surface of the metal oxide particles was achieved. Particle size distribution measurements revealed an average particle size of 495 d.nm for the functionalized material after the immobilization process. Quantitative determination of the BPCC shell content in TiO2-core:nano-Ca@ZOLE-shell-† was determined through thermogravimetric analysis, estimating a ratio of ∼1:3 (TiO2:BPCC). The cytotoxicity of TiO2-core:nano-Ca@ZOLE-shell-† 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 TiO2-core:nano-Ca@ZOLE-shell-† against MDA-MB-231, specifically at a concentration of 7.5 µM (% RCL = 46 ± 2%, 72 h). Under the same conditions, the functionalized material did not present cytotoxicity against hFOB 1.19 (% RCL ∼ 100%). These important outcomes provide evidence of the surface crystallization of BPCCs onto rutile phase TiO2 for the development of a novel functionalized material with the potential to treat and prevent osteolytic metastases.

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.

Potentiating bisphosphonate-based coordination complexes to treat osteolytic metastases.

The hydrothermal reaction between bioactive metal (Ca2+, Zn2+, and Mg2+) salts and a clinically utilized bisphosphonate, alendronate (ALEN), promotes the formation of several materials denominated as bisphosphonate-based coordination complexes (BPCCs). The systematic exploration of the effect of three variables, M2+/ALEN molar ratio, temperature, and pH, on the reaction yielded an unprecedented number of materials of enough crystal quality for structural elucidation. Five crystal structures were unveiled by single crystal X-ray diffraction (ALEN-Ca forms I and II, ALEN-Zn forms I and II, and ALEN-Mg) and their solid-state properties revealed in tandem with other techniques. The dissolution of these BPCCs was tested and contrasted to that of the commercially employed generic form of Fosamax® Alendronate Sodium, using fasted-state simulated gastric fluid and phosphate-buffered saline solution. Quantification of ALEN content was performed by derivatization with Cu2+, which produced a soluble complex suitable for quantification. The results show that these materials present a pH-dependent degradation. Moreover, a phase inversion temperature (PIT) nano-emulsion method was applied to the synthesis of ALEN-Ca form II. Size distribution analysis demonstrated the efficiency of the PIT-nano-emulsion method to decrease the particle size of this BPCC from ∼60 µm to ∼438 d nm. The cytotoxicity of ALEN, ALEN-Ca form II (bulk crystals), and nano-Ca@ALEN (nanocrystals) against the MDA-MB-231 cell line was investigated. Nano-Ca@ALEN form II presents higher cytotoxicity effects than ALEN and ALEN-Ca form II (bulk crystals), showing inhibition of cell proliferation at 7.5 µM. These results provide evidence of the structure, stability, dissolution and cytotoxicity properties of ALEN-based BPCCs and pave the way for better formulation strategies for this drug through the design of nano-sized BPCCs for the treatment of bone-related diseases.