Glutathione-Responsive Methotrexate Polymersomes for Potential Management of Ectopic Pregnancy

Abstract

The first-line treatment for ectopic pregnancy (EP), the chemotherapeutic methotrexate (MTX), has a failure rate of more than 10%, which can lead to severe complications or death. Inadequate accumulation of administered MTX at the ectopic implantation site significantly contributes to therapeutic failure. This study reports the first glutathione-responsive polymersomes for efficient delivery of MTX to the implantation site and its triggered release in placental cells. Fluorescence and photoacoustic imaging have confirmed that the developed polymersomes preferentially accumulate after systemic administration in the implantation site of pregnant mice at early gestational stages. The high concentrations of intracellular glutathione (GSH) reduce an incorporated disulfide bond within polymersomes upon internalization into placental cells, resulting in their disintegration and efficient drug release. Consequently, MTX delivered by polymersomes induces pregnancy demise in mice, as opposed to free MTX at the same dose regimen. To achieve the same therapeutic efficacy with free MTX, a sixfold increase in dosage is required. In addition, mice successfully conceive and birth healthy pups following a prior complete pregnancy demise induced by methotrexate polymersomes. Therefore, the developed MTX nanomedicine can potentially improve EP management and reduce associated mortality rates and related cost.

Keywords: ectopic pregnancy; glutathione; methotrexate; photoacoustic imaging; polymersome.

Figure 1.. Developed polymersomes and their characterization.

Schematic illustration of (A) MTX-SS-Ps (PEG-PCL disulfide bond-containing polymersome loaded with MTX), (B) MTX-Ps (polymersome lacking disulfide bond and loaded with MTX), and (C) NIR-SS-Ps (disulfide bond-containing polymersome loaded with NIR dye (silicon naphthalocyanine). Size distribution measured with dynamic light scattering (DLS) for (D) MTX-SS-Ps (38.2 nm ± 0.4, PDI: 0.11, n=3), (E) MTX-Ps (35.5 nm ± 0.3, PDI: 0.10, n=3), and (F) NIR-SS-Ps (39.3 nm ± 0.5, PDI: 0.12, n=3). Cryo-TEM images of (G) MTX-SS-Ps and (H) MTX-Ps. (I) Stability study via DLS size measurements for MTX-SS-Ps (red) and MTX-Ps (black) over 8 weeks. Drug release profiles of (J) MTX-SS-Ps in PBS (pH = 7.4) buffer containing 5 μM, 1 mM, and 10 mM GSH, and (K, L) MTX-SS-Ps (red) and MTX-Ps (black) at pH 7.4 (K) without GSH and (L) with 10 mM GSH.

Figure 2.. Biodistribution of NIR polymersomes in pregnant mice at Gd7.5.

Fluorescence images of (A) major organs and (B) mouse uteri, resected from pregnant mice (n=3) 24 hours post-injection (i.v.) at Gd6.5 with saline (control), NIR-Ps (0.3 mg mL⁻¹ dye, 100 μL) and NIR-SS-Ps (0.3 mg mL⁻¹ dye, 100 μL). (C) Semiquantitative analysis of fluorescence signal in 5 organs and uteri at Gd7.5, 24 hours post-injection with NIR-Ps, NIR-SS-Ps and saline (control). Values are expressed as mean ± SD (n=3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. H&E stained and corresponding fluorescence images of thin sections acquired at ex./em. 710/775nm of fetoplacental units from mice injected at Gd6.5 and collected 24 hours post-injection with (D) NIR-Ps (0.3 mg mL⁻¹ dye, 100 μL) and (E) NIR-SS-Ps (0.3 mg mL⁻¹ dye, 100 μL). Scale bar = 1 mm. am – antimesometrial decidua, md – mesometrial decidua, em – embryo, epc – ectoplacental cone, i.c. – implantation crypt, where em, epc, and i.c. are outlined in black.

Figure 3.. Photoacoustic imaging of placenta at Gd13.5 with NIR polymersomes.

Photoacoustic (PA) signal overlaid with ultrasound (US) images of ex vivo fetoplacental units, depicting NIR dye (green) and oxygenated hemoglobin (red), excised from pregnant mice (n=3) 24 hours following i.v. injection with (A) saline (control) and (B) NIR-SS-Ps (0.3 mg mL⁻¹ NIR dye, 100 μL) at Gd12.5 (Vevo LAZR imaging system equipped with an LZ550 transducer). Scale bar = 1 mm. PA signal spectra (averaged) corresponding to placental (black) and fetal (red) tissues from Figure 3A,B in pregnant mice treated with (C) saline and (D) NIR-SS-Ps. (E) H&E and (F) corresponding fluorescence images of thin section acquired at ex./em. 710/775 nm of a representative fetoplacental unit from mice injected with NIR-SS-Ps at Gd12.5 and collected 24 hours post-injection. Scale bar = 1 mm. a.c. – amniotic cavity, am – amnion, c.p. – chorionic plate, dec – decidua, em – embryo, j.z. – junctional zone, lab – labyrinth, mt – metrial triangle, um – umbilicus.

Figure 4.. Embryonic development following administration of MTX-SS-Ps and controls.

(A) Schematic timeline of treatment and imaging: MTX-SS-Ps (1 mg kg⁻¹) was i.v. injected at Gd6.5 and Gd8.5, concurrent with and followed by US monitoring through Gd13.5 (n=3). (B) Gestational sac length measurements acquired during US imaging. Values are expressed as mean ± SD (n=3). **P < 0.01, ***P < 0.001. (C) US images of embryos from each treatment group; MTX-SS-Ps and controls (saline, empty Ps, empty SS-Ps, free MTX and MTX-Ps (1 mg kg⁻¹ MTX at Gd6.5 and Gd8.5). Scale bar = 1 mm.

Figure 5.. Ex vivo evaluation of murine pregnancy progress following i.v. administration of free MTX, MTX-SS-Ps, MTX-Ps, and controls (saline and empty polymersomes).

Photographs of (A) exteriorized uteri and (B) resected uteri of mice (n=3) from Figure 4, euthanized at Gd13.5.

Figure 6.. Histological assessment of MTX nanomedicine treatment.

Top panels: H&E stained thin sections of selected fetoplacental units from Figure 5B: (A) saline, (B) free MTX, and (C) MTX-Ps, and (D) non-gravid uterus resulting from administration of MTX-SS-Ps. Scale bar: 1 mm. Bottom panels: Brightfield micrographs at 2.5x and 10x magnification of H&E stained histologic serial sections, from the same fetoplacental units depicted in the top panels, were investigated at different areas of the same section to analyze specific features. em – embryo, pl – placenta, end – endometrium, myo – myometrium, peri – perimetrium.

Figure 7.. Safety evaluation of MTX polymersomes to the mother and fetus, and subsequent pregnancy.

Changes in body weight of pups at post-natal day (pnd) 1 through pnd21 from (A) 1st pregnancy from each treatment group: control (saline, n=3), free MTX (1 mg kg⁻¹ 2x, n=3), and MTX-SS-Ps (1 mg kg⁻¹ 2x, n=3), and (B) subsequent 2nd pregnancy (without any treatment). (C) Changes in body weight of pups from treatment groups with various MTX doses: 2 mg kg⁻¹ 2x, 4 mg kg⁻¹ 2x, and 6 mg kg⁻¹ 2x.