PEGylated Polymeric Nanoparticles Loaded with 2-Methoxyestradiol for the Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model

Abstract

Leiomyomas, the most common benign neoplasms of the female reproductive tract, currently have limited medical treatment options. Drugs targeting estrogen/progesterone signaling are used, but side effects and limited efficacy in many cases are major limitation of their clinical use. Previous studies from our laboratory and others demonstrated that 2-methoxyestradiol (2-ME) is promising treatment for uterine fibroids. However, its poor bioavailability and rapid degradation hinder its development for clinical use. The objective of this study is to evaluate the in vivo effect of biodegradable and biocompatible 2-ME-loaded polymeric nanoparticles in a patient-derived leiomyoma xenograft mouse model. PEGylated poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles loaded with 2-ME were prepared by nanoprecipitation. Female 6-week age immunodeficient NOG (NOD/Shi-scid/IL-2Rγ^null) mice were used. Estrogen-progesterone pellets were implanted subcutaneously. Five days later, patient-derived human fibroid tumors were xenografted bilaterally subcutaneously. Engrafted mice were treated with 2-ME-loaded or blank (control) PEGylated nanoparticles. Nanoparticles were injected intraperitoneally and after 28 days of treatment, tumor volume was measured by caliper following hair removal, and tumors were removed and weighed. Up to 99.1% encapsulation efficiency was achieved, and the in vitro release profile showed minimal burst release, thus confirming the high encapsulation efficiency. In vivo administration of the 2-ME-loaded nanoparticles led to 51% growth inhibition of xenografted tumors compared to controls (P < 0.01). Thus, 2-ME-loaded nanoparticles may represent a novel approach for the treatment of uterine fibroids.

Keywords: Bioavailability; Drug delivery system; Estrogen; Fibroid; Nanoparticles; Precision medicine.

Figure 1.

Experimental design: (A) Schematic of the leiomyoma xenograft mouse model. (B) Administration paradigm for the 2-ME-loaded nanoparticles. (C) Representative histological fibroid image.

Figure 2.

In vitro drug release profile of 2-methoxyestradiol from PEGylated PLGA nanoparticles at 37°C in phosphate buffered saline (pH 7.4). Results are expressed as the mean ± s.d. (n=3).

Figure 3.

Differential scanning calorimetry thermographs of pure 2-methoxyestradiol, 2-methoxyestradiol-loaded nanoparticles, and blank (unloaded) nanoparticles. Samples were heated from 5°C to 215°C at a rate of 10°C per minute.

Figure 4.

Atomic force microscopy image of 2-methoxyestradiol-loaded PEGylated PLGA nanoparticles at 25°C.

Figure 5.

Cell viability of human uterine leiomyoma (huLM) cells treated with free 2-methoxyestradiol (2-ME) in solution, 2-methoxyestradiol-loaded nanoparticles (NPs), or blank (unloaded) nanoparticles at various concentrations of 2-methoxyestradiol, as measured by the WST-1 assay after treatment for 48 hours (A) or 72 hours (B). The blank nanoparticle treatments matched the polymer concentrations of the corresponding drug-loaded nanoparticle treatments. Triton-X 100 (0.1%, v/v) and cell culture medium served as negative and positive controls, respectively, with data presented as percent of control (cell culture media treatments set as 100%). Results are expressed as the mean ± s.d. (n=6). *, P < 0.05 in comparison to the media control. #, P < 0.05 in comparison to the free drug at the same concentration.

Figure 6.

Fibroid volume (A) and fibroid weight (B) after 28 days of treatment with 2-ME-loaded nanoparticles or control (unloaded nanoparticles). Fibroid volume was measured by caliper and fibroid weight was measured by postmortem scale in both groups. *, P < 0.05 by two-tailed unpaired t-test.

Figure 7.

Histological evaluation of fibroids from mice treated with control (unloaded nanoparticles) or 2-ME-loaded nanoparticles. Representative immunohistochemistry of (A) cyclin D1 expression, (B) Ki-67 expression, (C) progesterone receptor (PR) expression, and (D) estrogen receptor (ER) expression. (E) Quantification of marker expression between the control-treated and the 2-ME-treated groups. *, P < 0.05 by two-tailed unpaired t-test. Scale bars 20 μm. Number of immune-positive cells were counted and compared to total number of nuclei. per high power field.