A Nano-Platform for in situ Investigations of Ovarian Follicles

Abstract Background: Despite the growing array of assisted reproductive techniques, there is still a lack of rapid, non-cytotoxic, and minimally invasive in situ approaches for further enhancements through cell targeting. Here we synthesized clinically relevant liposome nanoparticles for real-time cellular targeting and drug (doxorubicin) delivery, using pigs and mares as animal models. In Experiment 1, fluorescently labeled and doxorubicin-loaded (without fluorescent probe) liposomes were injected in cultured pig ovarian follicles to assess plasma membrane binding and intracellular doxorubicin delivery. In Experiment 2, fluorescent liposomes were in vivo injected into small and large ovarian follicles of living mares to assess their binding capability to follicular cells. Twenty-four hours post-injection, all cultured pig follicles were collected while mare samples (i.e., follicle wall fragments, granulosa cells and follicular fluids) were harvested through follicle wall biopsy (FWB), and follicle aspiration and flushing techniques using transvaginal ultrasound-guided approach. Results: All injected follicles were healthy and samples were subjected to fluorescence imaging before and after fixation. Findings revealed successful intrafollicular migration and binding of liposomes to all follicle cell layers (granulosa, theca interna, and theca externa) regardless of the follicle size. The intracellular delivery of doxorubicin was confirmed with the staining of nuclei of follicle cells. Conclusions: This study demonstrates the promising combination of the FWB technique and nanotechnology tools for real-time monitoring of intrafollicular treatment, follicular health and oocyte development, which in turn has the potential to help understand the mechanisms of ovulatory dysfunction and to select high-quatity oocytes for assisted reproduction techniques.