Liposomal 2-Methoxyestradiol Nanoparticles for Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model

Abstract

Uterine leiomyomas represent a challenging problem with limited medical treatment options. The anti-tumor agent 2-methoxyestradiol (2-ME) shows promising results but its efficacy is limited by inadequate pharmacokinetics. We previously demonstrated that 2-ME nanoparticles can be successfully formulated and that they show improved in vitro anti-leiomyoma cell activity. Here, we examined the effects of the in vivo delivery of 2-ME nanoparticles in a patient-derived xenograft (PDX) leiomyoma mouse model. Patient-derived leiomyoma tumor tissues were xenografted subcutaneously in estrogen/progesterone pretreated immunodeficient NOG mice. Animals (n = 12) were treated with liposomal 2-ME nanoparticles by intra-peritoneal (IP) injection (50 mg/kg/dose, three times weekly) or control for 28 days. Tumor volume was measured weekly by calipers and prior to sacrifice by ultrasound. In addition, the expression of the cell proliferation marker Ki67 and the apoptosis marker cleaved caspase-3 in tumor tissues after treatment were measured by immunohistochemistry. Liposomal 2-ME treatment was associated with a significant tumor growth inhibition (30.5% less than controls as early as 2 weeks, p = 0.025). In addition, injections of liposomal 2-ME inhibited the expression of the proliferation marker Ki67 (55.8% reduction, p < 0.001). Furthermore, liposomal 2-ME treatment was associated with a 67.5% increase of cleaved caspase-3 expression of increase (p = 0.048). Our findings suggest that liposomal nanoparticle formulation can successfully deliver 2-ME and can be a promising therapeutic strategy for uterine leiomyoma. Further characterization of the liposomal-2ME, including pharmacokinetics, maximal tolerated dose, and safety, is needed in preclinical models prior to clinical trials.

Keywords: 2-ME; 2-methoxyestradiol; 2ME; Fibroid; Leiomyoma; Liposomes; Nanoparticles; Treatment.

Figure 1.

Mouse model. A: subcutaneous leiomyoma xenograft in the mouse as seen by ultrasound. B: leiomyoma xenograft as seen after mouse sacrifice.

Figure 2.

Effect of liposomal 2-ME nanoparticles treatment of patient-derived leiomyoma xenograft mouse model on tumor volume. Animals received 28 days of three times a week intraperitoneal injection (IP) of liposomal 2-ME containing 50 mg/kg mice body weight dissolved in 150μl saline (Liposomal 2-ME group, n=6) or equal volumes of empty vehicle (control group, n=6). Panel A: Tumor volume in mm³ as measured weekly by calipers. Panel B: Tumor size prior to sacrifice as measured by ultrasound. Data are presented as means ± SEM. (*) denotes statistical significance (p<0.05).

Figure 3.

Effect of liposomal 2-ME nanoparticles treatment of patient-derived leiomyoma xenograft mouse model on expression of Ki67. Animals were treated as described in Figure 1. Tumor tissues obtained after sacrifice were placed in 4% formalin solution and then paraffin sections were prepared. These sections were next used for immunostaining with the proliferation marker Ki67 with DAB as a chromogen. Expression was then graded by a blinded experienced pathologist using arbitrary units of expression assigned taking into account the tissue area, the percentage of positively stained cells to the total number of cells, and the intensity of the staining. This was performed in 10 separate high-power fields (20x) per slide. Graphic presentation of the expression of Ki67 in control and liposomal 2-ME groups (A: quantification; B: representative section). Data is presented as means ± SEM. (*) denotes statistical significance (p<0.05).

Figure 4.

Effect of liposomal 2-ME nanoparticles treatment of patient-derived leiomyoma xenograft mouse model on expression of cleaved caspase-3. Animals were treated as described in Figure 1. Immunohistochemistry, quantification and analysis for cleaved caspase-3 were performed as described in Figure 2. Graphic presentation of the expression of cleaved caspase-3 in control and liposomal 2-ME groups (A: quantification; B: representative section). Data is presented as means ± SEM. (*) denotes statistical significance (p<0.05).