Nano-encapsulation of arsenic trioxide enhances efficacy against murine lymphoma model while minimizing its impact on ovarian reserve in vitro and in vivo

Abstract

Advances in cancer therapy have increased the rate of survival of young cancer patients; however, female lymphoma patients frequently face a temporary or permanent loss of fertility when treated with traditional cytotoxic agents. The potential loss of fertility is an important concern that can influence treatment decisions for many premenopausal cancer patients. The negative effect of chemotherapeutic agents and treatment protocols to patients' fertility-referred to as fertotoxicity-are thus an increasingly important cancer survivorship issue. We have developed a novel nanoscale formulation of arsenic trioxide, a potent drug for treatment of hematological malignancies, and demonstrate that it has significantly better activity in a murine lymphoma model than the free drug. In parallel, we have developed a novel in vitro assay of ovarian follicle function that predicts in vivo ovarian toxicity of therapeutic agents. Our results reveal that the nanotherapeutic agent is not only more active against lymphoma, but is fertoprotective, i.e., it is much less deleterious to ovarian function than the parent drug. Thus, our in vitro assay allows rapid evaluation of both established and experimental anticancer drugs on ovarian reserve and can inform the selection of efficacious and fertility-sparing treatment regimens for reproductive-age women diagnosed with cancer.

Figure 1. NB(Ni,As) inhibits mantle cell lymphoma growth.

18 days after inoculation with Z138C lymphoma cells, Rag2M mice were randomized and treated with weekly injections of NB(NaCl), As₂O₃ (4, 6, or 8 mg/kg), or NB(Ni,As) (4, 6, or 8 mg/kg). (A) Tumors treated with NB(Ni,As) were significantly smaller than those treated with NB(NaCl). **, P>0.01, ***, P>0.001. Arrows indicate treatment. (B) Weight was monitored daily during the treatment period. Injection of As₂O₃ was acutely toxic, whereas mice injected with NB(NaCl) showed normal weight gain. Mice injected with any dose of NB(Ni,As) lost weight, though mice treated with 4 mg/kg showed the least amount of weight loss during the treatment period.

Figure 2. Arsenic plasma concentrations and uptake in mouse tissues and cultured mouse ovaries.

(A) NB(Ni,As)-treated (4 mg/kg) mice had reduced clearance of arsenic in plasma and increased peak plasma concentration compared with As₂O₃-treated (4 mg/kg) mice. (B) Arsenic levels in the uterus and ovaries peaked and cleared more rapidly in mice treated with As₂O₃ compared with mice treated with NB(Ni,As). (C) Arsenic levels in the liver and kidney paralleled those in the uterus. Error bars represent ± SEM.

Figure 3. Effect of As₂O₃ and NB(Ni,As) on ovarian cyclicity.

(A) All mice treated with PBS or NB(Ni,As) displayed normal cyclicity. Estrus cycles were stopped in 40% of mice treated with 4 mg/kg As₂O₃. One mouse treated with NB(NaCl) skipped one cycle, but otherwise cycled normally. Error bars represent ± SEM. (B) Representative cycles for each treatment group, [C, estrus, cornified epithelium present], [L, metestrus/diestrus, leukocytes present], [N, proestrus, nucleated cells present]. Arrows indicate treatment.

Figure 4. Effect of As₂O₃ and NB(Ni,As) on ovarian histology.

Hematoxylin and eosin staining of ovarian sections from mice following 3.5-weeks of treatment with (A) PBS (4× magnification); (B) 4 mg/kg NB(NaCl) 4×magnification); (C, E, F) 4 mg/kg As₂O₃ (4× and 10× magnification); or (D) 4 mg/kg NB(Ni,As) (4× magnification). (A, B, D) Ovaries from PBS-, NB(NaCl)-, and NB(Ni,As)-treated mice show normal ovarian histology and contain follicles of all stages as well as corpora lutea. (C, E, F) Ovaries isolated from As₂O₃-treated mice contained blood filled cysts and leaky vasculature. Measurement bars represent 100 µm (A–D) and 200 µm (E, F). Follicles are indicated with arrowheads and corpora lutea are labeled “CL.” Blood-filled cysts are indicated with arrows and areas of leaky vasculature are labeled “Bl” in panels C, E, and F.

Figure 5. Follicle survival after in vitro arsenic exposure.

Isolated ovarian follicles were incubated in PBS or (A) 3, (B) 30, or (C) 90 µM As₂O₃, NB(NaCl), or NB(Ni,As) for 3 hours. Individual follicles were then encapsulated in alginate and cultured for 10 days to analyze survival rate. (A) At 3 µM As₂O₃, follicle survival was not statistically significantly different compared with PBS, NB(NaCl), or NB(Ni,As). (B) At 30 µM As₂O₃, follicle survival was significantly less starting at day 4. C, At 90 µM As₂O₃, follicle survival dropped to 30% by day 4. At all concentrations, NB(Ni,As)-treated follicle survival was not significantly different than that of PBS or NB(Ni,As). (D) Ovaries were incubated in PBS or 3, 30, or 90 µM As₂O₃, NB(NaCl), or NB(Ni,As) for 3 hours. Arsenic content in the cultured ovaries was examined by ICP-MS. Arsenic content was significantly higher in As₂O₃-treated ovaries than in PBS-, NB(NaCl)-, or NB(Ni,As)-treated ovaries at 30 and 90 µM. Arsenic content in NB(NaCl)-treated ovaries was only significantly less than in ovaries treated with the highest dose of NB(Ni,As) (90 µM). (a) is P<0.01 compared with PBS, (b) is P<0.01 compared with NB(Ni,As) at the same concentration, (c) is P<0.01 compared with NB(NaCl) at the same concentration. Error bars represent ±SEM.

Figure 6. Follicle growth after in vitro arsenic exposure.

Isolated ovarian follicles were incubated in PBS or (A) 3, (B) 30, or (C) 90 µM As₂O₃, NB(NaCl), or NB(Ni,As) for 3 hours. Individual follicles were then encapsulated in alginate and cultured for 10 days to analyze follicle growth. (A, B) At 3 and 30 µM, all surviving follicles grew to approximately the same size, between 200 and 250 µm. (C) At 90 µM, As₂O₃-treated follicles showed a decrease in follicle diameter over 10 days of culture, producing significantly smaller follicles than those treated with NB(NaCl) or NB(Ni,As). Error bars represent ± SEM. Asterisk represents significance of P<0.05.