Bisphosphonate-Functionalized Hydroxyapatite Nanoparticles for the Delivery of the Bromodomain Inhibitor JQ1 in the Treatment of Osteosarcoma

Abstract

Osteosarcoma (OS) is one of the most common neoplasia among children, and its survival statistics have been stagnating since the combinatorial anticancer therapy triad was first introduced. Here, we report on the assessment of the effect of hydroxyapatite (HAp) nanoparticles loaded with medronate, the simplest bisphosphonate, as a bone-targeting agent and JQ1, a small-molecule bromodomain inhibitor, as a chemotherapeutic in different 2D and 3D K7M2 OS in vitro models. Both additives decreased the crystallinity of HAp, but the effect was more intense for medronate because of its higher affinity for HAp. As the result of PO43−-NH+ binding, JQ1 shielded the surface phosphates of HAp and pushed its surface charge to more positive values, exhibiting the opposite effect from calcium-blocking medronate. In contrast to the faster and more exponential release of JQ1 from monetite, its release from HAp nanoparticles followed a zero-order kinetics, but 98% of the payload was released after 48 h. The apoptotic effect of HAp nanoparticles loaded with JQ1, with medronate and with both JQ1 and medronate, was selective in 2D culture: pronounced against the OS cells and nonexistent against the healthy fibroblasts. While OS cell invasion was significantly inhibited by all of the JQ1-containing HAp formulations, that is, with and without medronate, all of the combinations of the targeting compound, medronate, and the chemotherapeutic, JQ1, delivered using HAp, but not HAp alone, inhibited OS cell migration from the tumor spheroids. JQ1 delivered using HAp had an effect on tumor migration, invasion, and apoptosis even at extremely low, subnanomolar concentrations, at which no effect of JQ1 per se was observed, meaning that this form of delivery could help achieve a multifold increase of this drug’s efficacy. More than 80% of OS cells internalized JQ1-loaded HAp nanoparticles after 24 h of coincubation, suggesting that this augmentation of the activity of JQ1 may be due to the intracellular delivery and sustained release of the drug enabled by HAp. In addition to the reduction of the OS cell viability, the reduction of the migration and invasion radii was observed in OS tumor spheroids challenged with even JQ1-free medronate-functionalized HAp nanoparticles, demonstrating a definite anticancer activity of medronate alone when combined with HAp. The effect of medronate-functionalized JQ1-loaded HAp nanoparticles was most noticeable against OS cells differentiated into an osteoblastic lineage, in which case they surpassed in effect pure JQ1 and medronate-free compositions. The activity of JQ1 was mediated through increased Ezrin expression and decreased RUNX2 expression and was MYC and FOSL1 independent, but these patterns of gene expression changed in cells challenged with the nanoparticulate form of delivery, having been accompanied by the upregulation of RUNX2 and downregulation of Ezrin in OS cells treated with medronate-functionalized JQ1-loaded HAp nanoparticles.

Nanoparticles Loaded with the BET Inhibitor JQ1 Block the Growth of Triple Negative Breast Cancer Cells In Vitro and In Vivo

“…Various other formulations have been developed in order to entrap JQ1; bisphosphonate-functionalized hydroxyapatite-, phospholipid-and chitosan-based nanostructures containing the active compound have been characterized and proposed for the treatment of various diseases, including osteosarcoma, glioma and liver fibrosis, respectively [37][38][39]. Biomimetic nanoclusters made up of the components of the platelet membrane and polyamidoamine-polyvalerolactone-COOH (PAMAM-PVL-COOH) have also been described as a potential endothelium-protective anti-restenotic system, improving the peculiar pharmacological properties of JQ1 [40].…”

Three‐Dimensional Osteosarcoma Models for Advancing Drug Discovery and Development

“…[75] Similar to scaffold-free models, scaffold-based ones are used to study CSCs resistance mechanisms and search for new therapeutic agents. [77] The 2D assays demonstrated a promising selectivity of HAp/BP/JQ1 nanoparticles toward cancer cells, having no toxic effect on cell viability and migration of primary fibroblasts. [76] rBM-based hydrogels were also applied in spheroid invasion models to evaluate the pharmacodynamic effect of medronate (BP)-functionalized HAp nanoparticles loaded with the bromodomain inhibitor JQ1 on OS cancer cells.…”

“…[76] rBM-based hydrogels were also applied in spheroid invasion models to evaluate the pharmacodynamic effect of medronate (BP)-functionalized HAp nanoparticles loaded with the bromodomain inhibitor JQ1 on OS cancer cells. [77] The 2D assays demonstrated a promising selectivity of HAp/BP/JQ1 nanoparticles toward cancer cells, having no toxic effect on cell viability and migration of primary fibroblasts. Regarding OS cell invasion, fabricated nanoparticles also significantly inhibited OS spheroid invasion.…”

“…Matrigel Hydrogel HOS, 143B -pLKO.1 shsFRP2 vector [73] 143B, MG-63, U2OS, hFOB1.19 -Paris polyphylla ethanol extract [75] MG-63 -miR-29b-1 overexpression [76] K7M2 -Bromodomain inhibitor JQ1 [77] Collagen Hydrogel U2OS -Kinase inhibitor PI103 [43] Type I MG-63 -Tyrosin phosphorylation inhibitor (ACM-14) [91] LM8 -Pazopanib [96] Sponge hFOB 1.19 (osteoblasts) PC3, LNCaP siRNA-based gene knockdown [97] Matrigel/Collagen Hydrogel MOS, U2OS, 143B, ZK58, KPD, SaOS-2 Trametinib, AZD8330, and TAK-733 kinase inhibitors [79] MOS, U2OS, 143B, ZK58, KPD -siAven [78] Silk Fibroin Sponge 143.98.2, SaOS-2, U2OS -Doxorubicin, cisplatin [108] U2OS Bone marrow fibroblasts, HUVECs MAB208 (anti-IL-8), Avastin (anti-VEGF-A) [109] MG-63 MDA-MB-231 Doxorubicin [110] MG-63 MDA-MB-231, MSCs Paclitaxel [111] Chitosan Sponge MG-63, MNNG -Doxorubicin [139] Alginate Hydrogel MG-63 -Tetracycline hydrochloride [115] Fibers MG-63 HUVECs - [116] Spheres Dunn, LM8 NF-кB decoy oligodeoxy-nucleotide [124] Synthetic Biomaterials…”

“…Reproduced with permission [77]. A) Schematic illustration of HAp nanoparticles and its role in OS treatment.…”