A novel nanoparticulate formulation of arsenic trioxide with enhanced therapeutic efficacy in a murine model of breast cancer

Abstract

Purpose: The clinical success of arsenic trioxide (As(2)O(3)) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As(2)O(3) encapsulated in liposomal vesicles or "nanobins" [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As(2)O(3) would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas.

Experimental design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As(2)O(3) was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As(2)O(3) were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer.

Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As(2)O(3) against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As(2)O(3) in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As).

Conclusions: Nanobin encapsulation of As(2)O(3) improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors.

Figure 1

NB(Ni,As) is a novel formulation of As₂O₃ in which the active drug is encapsulated as a nanoparticulate inside a lipid bilayer. A, schematic representation of transition metal triggered precipitation of As₂O₃ inside the nanobin. B, transmission electron micrograph of NB(Ni,As) stained with 4% uranyl acetate shows homogenous particles ~100 nm in size (Scale Bar, 100 nm). C, kinetics of arsenic release in 80% FBS at 37°C are accelerated at pH 5.5 compared with pH 7.4. Points, mean; bars, ± SEM.

Figure 2

NB(Ni,As) attenuates As₂O₃ cytotoxicity in vitro. Breast cancer cells were treated with free As₂O₃ (■), NB(Ni,As) (▼), or NB(NaCl) (●). Cell viability was evaluated at 72 h by MTS assay. Points, mean; bars, ± SEM.

Figure 3

As₂O₃ and NB(Ni,As) induce apoptosis and inhibit migration and invasion in MDA-MB-231 triple negative breast cancer cells in vitro. A, MDA-MB-231-mCherry cells were treated with As₂O₃ or NB(Ni,As) ([As] = 50 µM. [As₂O₃] = 25 µM) , alone or in combination with pan-caspase inhibitor Z-VAD-FMK (50 µM). Apoptosis was measured by Annexin V labeling and membrane integrity was measured by DAPI by FACS analysis at 48 h. B, Migration was assessed by seeding MDA-MB-231-mCherry cells on the top of an uncoated transwell, and cells were treated with As₂O₃ (0.1, 1.0, 10.0 µM As), NB(Ni,As) (0.1, 1.0, 10.0 µM As) or NB(NaCl) control for 24 h. C MDA-MB-231-mCherry cells were seeded on Matrigel coated transwells, treated with with As₂O₃ (0.1, 1.0, 10.0 µM As), NB(Ni,As) (0.1, 1.0, 10.0 µM As) or NB(NaCl) for 48 h, and invading cells were scored. Bars ± SEM *P < 0.05; **P < 0.01, ***P<0.001 versus media control.

Figure 4

The pharmacokinetic properties of NB(Ni,As) and free As₂O₃ were measured in double jugular catheterized SD Rats (n=5). Rats received a single injection of NB(Ni,As) (4 mg As/kg) or free As₂O₃ (4 mg As/kg) and plasma samples were collected over a 24 h period. Points, mean; bars, ± SEM.

Figure 5

NB(Ni,As) inhibits mammary tumor growth in vivo in an orthotopic model of breast cancer. A, twelve days after orthotopic inoculation of MDA-MB-231-mCherry cells, female athymic nude mice were randomized into 4 treatment groups (8 mice per group): PBS, empty nanobins (NB(NaCl)), free As₂O₃ (4 mg/kg), and NB(Ni,As) (4 mg/kg). Agents were delivered twice weekly for 3 weeks by intraperitoneal injection as indicated. Mammary tumor volumes were measured using calipers (left panel). *P < 0.05; **P < 0.01 versus vehicle treated mice. mCherry fluorescence of mammary tumors at week 5.5 (right panels). B, enhanced arsenic accumulation in mammary tumors from mice treated with NB(Ni,As) compared to those receiving free As₂O₃. Tumor arsenic concentrations were measured by ICP-MS analysis in mice receiving 3 (3 Tx) or 5 doses (5 Tx) of these agents as indicated in A. **P < 0.01 versus NB(NaCl) control. C, immunohistochemical staining for active cleaved caspase-3 in mammary tumors from mice treated with PBS, NB(NaCl) NB(Ni,As) or free As₂O₃ for 2 weeks (5 doses). *P < 0.05; ***P < 0.001 versus NB(NaCl) treated mice. D, no significant weight loss was observed in any treatment group compared with vehicle treated mice.