Folate-targeted pH-responsive calcium zoledronate nanoscale metal-organic frameworks: Turning a bone antiresorptive agent into an anticancer therapeutic

Abstract

Zoledronate (Zol) is a third-generation bisphosphonate that is widely used as an anti-resorptive agent for the treatment of cancer bone metastasis. While there is preclinical data indicating that bisphosphonates such as Zol have direct cytotoxic effects on cancer cells, such effect has not been firmly established in the clinical setting. This is likely due to the rapid absorption of bisphosphonates by the skeleton after intravenous (i.v.) administration. Herein, we report the reformulation of Zol using nanotechnology and evaluation of this novel nanoscale metal-organic frameworks (nMOFs) formulation of Zol as an anticancer agent. The nMOF formulation is comprised of a calcium zoledronate (CaZol) core and a polyethylene glycol (PEG) surface. To preferentially deliver CaZol nMOFs to tumors as well as facilitate cellular uptake of Zol, we incorporated folate (Fol)-targeted ligands on the nMOFs. The folate receptor (FR) is known to be overexpressed in several tumor types, including head-and-neck, prostate, and non-small cell lung cancers. We demonstrated that these targeted CaZol nMOFs possess excellent chemical and colloidal stability in physiological conditions. The release of encapsulated Zol from the nMOFs occurs in the mid-endosomes during nMOF endocytosis. In vitro toxicity studies demonstrated that Fol-targeted CaZol nMOFs are more efficient than small molecule Zol in inhibiting cell proliferation and inducing apoptosis in FR-overexpressing H460 non-small cell lung and PC3 prostate cancer cells. Our findings were further validated in vivo using mouse xenograft models of H460 and PC3. We demonstrated that Fol-targeted CaZol nMOFs are effective anticancer agents and increase the direct antitumor activity of Zol by 80-85% in vivo through inhibition of tumor neovasculature, and inhibiting cell proliferation and inducing apoptosis.

Keywords: Cancer; Chemotherapy; Drug delivery; Folate-targeted nanoscale metal-organic frameworks; Zoledronate.

Fig. 1

Synthesis of Fol-targeted CaZol nMOFs. Hydrophobic DOPA-coated CaZol nMOFs were prepared via the water-in-oil microemulsion method. The hydrophilic Fol-targeted lipid-coating was fabricated onto the hydrophobic DOPA-coated CaZol nMOFs via the film rehydration method. Table S4 shows the chemical structures of lipids in the inner and outer lipid leaflets of the Fol-targeted CaZol nMOFs.

Fig. 2

Characterization of hydrophobic DOPA-coated CaZol nMOFs. (a) TEM images recorded for DOPA-coated CaZol nMOFs prepared using oils with different volume ratios of Igepal-based and Triton-based oil systems. The Igepal-based oil system is composed of a 71:29 v/v of cyclohexane and Igepal CO-520. The Triton-based oil is composed of a 75:15:10 v/v/v of cyclohexane, Triton X-100, and 1-hexanol. The diameters of the CaZol nMOFs increase as the volume fraction of the Triton-based oil phase increases. The pink arrow highlights the small, irregularly shaped CaZol nMOFs. (b) TGA curves recorded for (i) DOPA, (ii) CaZol bulk powder, and (iii) DOPA-coated CaZol nMOFs. It was calculated that the DOPA-coated CaZol nMOFs contained 12.7wt% of garfted DOPA.^‡ (c) An energy-dispersive X-ray spectrum recorded for DOPA-coated CaZol nMOFs. The inset table summarizes the bulk composition of the DOPA-coated CaZol nMOFs. (d) (i) XPS survey spectrum recorded for DOPA-coated CaZol nMOFs. The inset table summarizes the surface composition of the DOPA-coated nMOFs. (N.B. ^# the C and N bands are affected by the background of carbon tape used to hold the powders for the EDS study.) (ii) P 2p core-line spectrum recorded for the DOPA-coated CaZol nMOFs. The strong P 2p band can be attributed to the P atoms in the DOPA (stabilizer) and Zol (core of the nMOF). (iii) N 1s core-line spectrum recorded for the DOPA-coated CaZol nMOFs. The strong N 1s band can be attributed to the N atom in Zol's imidazole ring. (e) Assigned ATR FT-IR spectra recorded for (i) DOPA, (ii) deprotonated Zol (Na₄Zol), and (iii) dried DOPA-coated CaZol nMOFs. [N.B. ^‡ By comparing the wt% of bulk CaZol and DOPA-coated CaZol nMOFs remained at 800°C, the amount of grafted DOPA in the DOPA-coated CaZol nMOFs = 100wt% – (wt% of the DOPA-coated CaZol nMOFs remained at 800°C)/(wt% of the bulk CaZol MOFs remained at 800°C) = 100wt% - (100.0wt% - 41.4wt%)/(100.0wt% - 32.9wt%) = 12.7 wt %]

Fig. 3

Characterization of Fol-targeted PEGylated CaZol nMOFs. (a) TEM images recorded for (i) asymmetric lipid-coated Fol-targeted CaZol nMOFs and (ii) Fol-targeted liposomes prepared in the absence of DOPA-coated CaZol nMOFs. (b) Number-average diameter (D_n) distribution curves recorded for 5 μg/mL of (i) Fol-targeted CaZol nMOFs and (ii) Fol-targeted liposomes prepared in the absence DOPA-coated CaZol nMOFs, as determined by the NTA method. Calculations showed that 5 μg/mL of the Fol-targeted nMOFs contain 5.2 × 10⁹ particles per mL and that 5 μg/mL of Fol-targeted liposomes contain 2.3 × 10⁹ particles per mL. Thus, the average weight of each Fol-targeted CaZol nMOF is 9.6 × 10^-16 g, and the average weight of each Fol-targeted liposome is 2.2 × 10^-15 g. (c) Intensity-average diameter (D_h) distribution curves recorded for (i) DOPA-coated CaZol nMOFs (dispersed in chloroform), (ii) Fol-targeted CaZol nMOFs, and (iii) Fol-targeted liposomes prepared in the absence of DOPA-coated CaZol nMOFs. (d) TGA curves recorded for (i) DOPA-coated CaZol nMOFs, (ii) DOTAP, (iii) cholesterol (Chol), (iv) DSPE-PEG, and (v) asymmetric, lipid-coated Fol-targeted CaZol nMOFs. It was calculated that the Fol-targeted CaZol nMOFs contained 32.3wt% of outer lipid coating.^‡ [N.B. ^‡ By comparing the wt% of DOPA-coated CaZol NPs and Fol-targeted CaZol nMOFs remained at 800°C, the amount of outer Fol-targeted lipid coating in the Fol-targted CaZol nMOF = 100wt% – (wt% of the Fol-targeted CaZol nMOFs remained at 800°C)/(wt% of the Fol the DOPA-coated CaZol nMOFs remained at 800°C) = 100wt% – (100.0wt% -60.3wt%)/(100.0wt% - 41.4wt%) = 32.3wt%]

Fig. 4

Drug-release mechanism of Fol-targeted CaZol nMOFs. (a) In vitro drug-release kinetics of Fol-targeted CaZol nMOFs under sink conditions at pH 7.0 and pH 5.0 (in 0.1 M PBS at 37 °C). Insert shows chemical structure of deprotonated Zol in CaZol nMOF. pKa of Zol = 5.9, 8.3[35]. (b) Fluorescence images of Fura-2-loaded PC3 cells recorded (i) immediately, (ii) 30 min, and (iii) 2 h after being incubated with 1 μM of Fol-targeted CaZol nMOFs; and recorded (iv) after incubation in 0.1 M PBS for 2 h (control) at an excitation wavelength of 362 nm and an emission wavelength of 512 nm (the red channel, which is proportional to the concentration of free Fura-2) and an excitation wavelength of 362 nm and an emission wavelength of 512 nm (the green channel, which is proportional to the concentration of the Fura-2-Ca complex due to the Ca²⁺ released from the CaZol nMOFs). Bar chart summarizing the ratio of fluorescence intensities at 478 nm and 505 nm, which is proportional to the concentration of unbound Fura-2. (Fig. S3 shows the intracellular release of free Ca²⁺ ions from Fol-targeted CaZol nMOFs in the H460 lung-cancer cell line.) (c) Cartoon shows the cellular uptake, intercellular Ca²⁺ and Zol release mechanism of CaZol nMOFs and the formation of the Fura-2-Ca complex.

Fig. 5

In vitro toxicities of small-molecule (“free”) Zol, non-targeted and Fol-targeted CaZol nMOFs. Cell viabilities of (a) H460 and (b) PC3 cells after treatment with different concentrations of (i) drug-free Fol-targeted liposomes, (ii) small-molecule Zol, (iii) non-targeted CaZol nMOFs, and (iv) Fol-targeted CaZol nMOFs, as determined by a MTS cell-proliferation assay. (c) Early-stage apoptosis in H460 and PC3 cells. Confocal fluorescence images of (i) H460 and (ii) PC3 cells recorded after treatment with 5 μM of free Zol, NBD-labeled non-targeted CaZol nMOFs and Fol-targeted CaZol nMOFs containing 5 μM of encapsulated Zol for 2 h and incubation under physiological conditions for another 12 h before staining with Rhod-labeled caspase 3 (CASP 3, which is red fluorescent and an early-stage apoptosis marker). Fig. S6 shows confocal fluorescence images recorded for H460 and PC3 cells treated with 0.1 M PBS (control). (N.B. ^# containing the same amount of lipid coating as in the Fol-targeted CaZol nMOFs; e.g., 1 μM encapsulated Zol = 0.27 μg/mL of Fol-targeted CaZol nMOFs because Zol makes up 50.6 wt% of each Fol-targeted CaZol nMOF; the weight of a Fol-targeted CaZol nMOF is 9.6 × 10^-16 g, and the weight of each drug-free Fol-targeted liposome is 2.2 × 10^-15 g; and 1 μM of encapsulated Zol in a CaZol nMOF contains 1.2 μg/mL of Fol-targeted lipid coating; * p < 0.05, which is statistically significant.)

Fig. 6. In vivo

antitumor efficiencies of drug-free Fol-targeted liposomes, small-molecule Zol, and non-targeted and Fol-targeted CaZol nMOFs. (a)(i) Tumor growth-delay curves and (ii) Kaplan-Meier survival curves of H460 lung xenograft tumor-bearing mice after a single i.v. administration of PBS, drug-free Fol-targeted liposomes,^# or 0.8 mg/kg (half of the MTD of Zol in mice) of small-molecule or encapsulated Zol. (b)(i) Tumor growth-delay curves and (ii) Kaplan-Meier survival curves of PC3 prostate xenograft tumor-bearing mice after a single i.v. administration of PBS, drug-free Fol-targeted liposomes, or 0.8 mg/kg (half of the MTD of Zol in mice) of small-molecule or encapsulated Zol. (c) Table comparing in vivo antitumor efficiencies of small-molecule and encapsulated Zol. (N.B. ^# containing the same amount of lipid coating as the Fol-targeted CaZol nMOFs; i.e., each 29–30 g mouse received IV administration of 200 μL of 245μg/mL of drug-free Fol-targeted liposomes; n.s. = statistically insignificant; * p < 0.05, which is statistically significant.)

Fig. 7

Histology of H460 and PC3 tumor sections after received different Zol treatments. (a) CD31-stained H460 and PC3 tumor sections after treated with PBS, small molecule (“free”) Zol, non-targeted and Fol-targeted CaZol nMOFs. The CD31 antibody (brown) labeled the vascular endothelial cells. (b) PCNA- and (c) caspase 3-stained H460 and PC3 tumor sections after treatment with PBS, small-molecule (“free”) Zol, or non-targeted and Fol-targeted CaZol nMOFs. The PCNA antibody (red fluorescence) indicates a nucleus undergoing cell proliferation. The Caspase 3 antibody (red fluorescence) indicates cells undergoing early-stage apoptosis (programmed cell death). (N.B. the nuclei in all tumor sections were co-stained with DAPI (blue fluorescence); * p < 0.05, which is statistically significant.)

Fig. 8

Direct anticancer activity of Fol-targeted CaZol nMOFs against a Fol-receptor-overexpressed cancer cell. Active-targeted PEGylated CaZol nMOFs bind to the Fol receptor in Fol-receptor-overexpressed cancer cells and enter the cells via endocytosis. The (partial) protonation of CaZol nMOFs in the mildly acidic endosomes (pH ≈ 5) triggers the release of therapeutic, active Zol. Zol inversely inhibits farnesyl diphosphate synthase (FPPS), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (vEGF), which in turn inhibit cancer-cell proliferation, trigger apoptosis, and inhibit blood-vessel growth. (N.B. HMG-CoA = 3-hydroxy-3-methylglutaryl-coenzyme A; IPP = isopentenyl pyrophosphate; GPP = geranyl pyrophosphate; FPP = farnesyl pyrophosphate; GGPP = geranylgeranyl pyrophosphate)