Nanoparticles Accumulate in the Female Reproductive System during Ovulation Affecting Cancer Treatment and Fertility

Abstract

Throughout the female menstrual cycle, physiological changes occur that affect the biodistribution of nanoparticles within the reproductive system. We demonstrate a 2-fold increase in nanoparticle accumulation in murine ovaries and uterus during ovulation, compared to the nonovulatory stage, following intravenous administration. This biodistribution pattern had positive or negative effects when drug-loaded nanoparticles, sized 100 nm or smaller, were used to treat different cancers. For example, treating ovarian cancer with nanomedicines during mouse ovulation resulted in higher drug accumulation in the ovaries, improving therapeutic efficacy. Conversely, treating breast cancer during ovulation, led to reduced therapeutic efficacy, due to enhanced nanoparticle accumulation in the reproductive system rather than at the tumor site. Moreover, chemotherapeutic nanoparticles administered during ovulation increased ovarian toxicity and decreased fertility compared to the free drug. The menstrual cycle should be accounted for when designing and implementing nanomedicines for females.

Keywords: breast cancer; fertility; gender medicine; gold nanoparticles; liposome; mRNA LNP; ovarian cancer.

Figure 1. Biodistribution of nanoparticles to the reproductive system during the female mouse menstrual cycle.

During the estrus stage there is increased blood supply to the ovary to support preovulatory follicles. After ovulation, a dense blood network termed the corpus luteum is observed. Higher density of blood vessels around the follicle result in higher accumulation of nanoparticles (blue) in the reproductive system (a). Contrarily, there are fewer blood vessels in the ovary and around the follicles specifically during the diestrus stage (b). 80-nm Gd-loaded PEGylated liposomes (Gd-lipo) were imaged using cryo-TEM (c, scale bar - 100 nm) and sized using dynamic light scattering (DLS) (d). Gd-lipo were injected intravenously (i.v.) to female mice at different stages of the menstrual cycle (e). Nanoparticle’ accumulation 24-hours post administration was quantified using elemental analysis for Gd, or by mRNA expression. Results are shown as the injected %Gd normalized to the organ weight. 1.8-fold more liposomes reached the ovaries at the estrus stage (n=8) compared to the diestrus stage (n=8,red and blue represent two independent experiments) (f). 2.5-fold more liposomes reached the uterus at the estrus stage (n=8) compared to at the diestrus stage (n=7) (g). Ex-vivo fluorescent images of the female murine reproductive system 24-hours post i.v. injection of 80-nm Cy5-labeled liposomes during the different ovulation stages (h, scale bar - 0.5 cm). Blood vessel density was evaluated using anti-CD31 immunohistochemistry staining at diestrus (i) and estrus stages (j) (scale bar - 100 μm) and was quantified as the percentage of stained area compared to a control stained only with secondary Ab (k). Expression of firefly-luciferase in the ovaries during estrus, 24-hours post i.v. injection of lipid nanoparticles loaded with firefly-luciferase mRNA. The bottom image is a control without nanoparticles injection (scale bar 0.5 cm) (l). Results are shown as mean±SEM. One-way ANOVA and Tukey’s t-test were used for statistical analysis of f and g, unpaired two-tail t-test was used for statistical analysis of k. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Illustrations e, f and g were made using BioRender.

Figure 2. Size-dependence and nanoparticle biodistribution to the ovaries, uterus and other organs.

The biodistribution of Gd-lipo to the (a) heart, kidneys, liver, lung and spleen at the different stages of the estrus cycle, and (b) to the adrenal glands at estrus and diestrus. Size-dependent accumulation of gold nanoparticles in the ovaries (blue circles) and the uterus (red squares) 24-hours after i.v. injection during the estrus stage (n=4 for all groups) (c). 80-nm liposomes were detected at the blood-follicle barrier as demonstrated by fluorescent histology images of Cy5-labeled liposomes (pink) localization in the ovary (nuclei - blue) 24-hours after i.v. injection (d, scale bar - 100 μm). Line profile of the fluorescent intensity signal across a single follicle shows that the liposomes surround the follicle, indicated by two peaks in the dye signal (e). Illustration of the blood-follicle barrier shows the liposomes (blue) on the basal membrane of the follicle and are restricted to the thecal layer around the follicle (f). Results are shown as mean±SEM. Unpaired two-tail t-test was used for statistical analysis of a. One-way ANOVA and Tukey’s t-test were used for statistical analysis of b and c. *p<0.05, **p<0.01.

Figure 3. Biodistribution of liposomes to breast and ovarian cancer tumors, and the efficacy of cancer treatment are affected by the female mouse cycle.

During the estrus stage, Gd-liposomes accumulate in the reproductive system (n=4) at higher levels than in orthotopic triple-negative murine (4T1) breast cancer tumors(n=4). In contrast, during the diestrus stage nanoparticles shift towards the tumor (n=5) and away from the reproductive system (n=5) (a). Maximum intensity projection (MIP) SPECT/CT images of an MDA-MB-231 breast cancer tumor-bearing mouse after i.v. injection of ¹¹¹In-lipo. Accumulation is detected in the liver (1), spleen (2), ovaries (3), uterus (3*) and the tumor (4), 48-hours (right image) and 7 days (left image) after injection (b). In mice bearing orthotopic ovarian cancer in one of the two ovaries, there is increased accumulation of Gd-liposomes both in the tumor-bearing ovary (Estrus n=6, Diestrus n=8) and in the healthy ovary (Estrus n=6, Diestrus n=7) during the estrus stage, compared to the diestrus stages (c). Efficacy of DOX-lipo was evaluated using caliper measurements in 4T1 mCherry breast cancer model during estrus (n=7) and diestrus (n=6). Arrows indicate treatment times (d). IVIS images of 4T1 mCherry breast cancer tumor (top - treatment during estrus, bottom - treatment during diestrus) show 3 representative mice from each group (e). For ovarian cancer, treatment efficacy of DOX-lipo was evaluated using IVIS imaging, enabled by the luciferase-expressing cells, during the estrus (n=5 (days 0 and 7), n=2 (day 14)) and diestrus stages (n=5 (days 0 and 7), n=3 (day 14)). Arrows indicate treatment times (f). IVIS images of luminescent ovarian cancer tumor, left panel - treatment during diestrus, right panel - treatment during estrus (g). Results are shown as mean±SEM. Two-way ANOVA and Tukey’s t-test were used for statistical analysis of a-c, e. **p<0.01, ***p<0.001, ****p<0.0001. Images a and b were created using BioRender.

Figure 4. Doxorubicin-loaded liposomes delayed ovarian toxicity and affected fertility in female mice.

Healthy female mice received an i.v. injection of either free DOX or DOX-lipo, and the ovaries were analyzed by immunohistochemistry and RT-PCR 24- and 48-hours post drug administration (a). RT-PCR of pro/anti apoptotic gene expression (Bcl2 and BAX) during estrus (24-hours, n=3 for free DOX n=5 for DOX-lipo. 48-hours, n=3 for both groups) and diestrus (24-hours, n=3 for free DOX n=4 for DOX-lipo. 48-hours, n=3 for both groups) are shown as the relative expression of the ratio between pro- (BAX) and anti-apoptotic (bcl2) genes (b). Immunohistochemistry of anti-active caspase3 24-hours after i.v. injection during the estrus stage of either free DOX (c) or DOX-lipo (d) show apoptotic follicles (brown signal) with higher signal in the follicles in the free DOX group. Quantification of the total signal output from the follicles demonstrated that more follicles were apoptotic in the free DOX group (n=4) compared to DOX-lipo (n=5) 24-hours after i.v. injection, however apoptosis levels become comparable after 48-hours (n=5 for both groups) (e). Healthy female mice received repeated i.v. injections of either free DOX (n=10) or DOX-lipo (n=10) once-a-week for 3 consecutive treatments and their cycle stage was recorded daily to be compared to that of the control group (n=9) that was not injected (f). The estrous cycle in each group is demonstrated in graphs g-i where each line represents a single mouse. Compared to the control group (g), mice in the DOX-lipo (h) and the free DOX (i) groups had a disrupted cycle. After the third injection, females were housed with males and the day of birth, litter size and pups’ viability were recorded (j). The time until pregnancy was significantly longer for both the free DOX and the DOX-lipo group, however the pups’ viability was lower for the DOX-lipo group. Results are shown as mean±SEM. Three-way ANOVA and Tukey’s t-test (b), two-way ANOVA and Tukey’s t-test (e and %alive pups in j) and Dunnett's T3 t-test (days until birth in j) were used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Images a and f were created using BioRender.