The current efforts to overcome fertility problems through assisted reproductive technologies (ARTs) are still unsatisfactory because of the insufficient maturation of the oocytes and/or the existing follicular diseases [[1], [2], [3], [4], [5], [6]]. Furthermore, the ability to assess the oocytes’ quality mainly, if not only, through in vitro manipulations constitutes a major limitation [7,8]. Therefore, developing novel and less invasive methods for intrafollicular assessment of oocyte quality or local interventions in unhealthy follicles (e.g., disease treatments) before retrieving enclosed oocytes would be of great interest. In this regard, various methodologies have been used to address the challenges related to female fertility treatments. For example, routine systemic drug injections (i.e., exogenous gonadotropins) for controlled ovarian hyperstimulation (COH) show increased evidence of detrimental effects on oogenesis, embryo quality, and endometrial receptivity [[9], [10], [11], [12]]. These negative effects are due to the uncontrollable doses of injected drugs reaching the target tissues or cells. Alternative treatments such as local or intrafollicular injection techniques (injection and flushing) have been applied in large animals (e.g., cow and mare) with relative success [4,13] as a better-controlled route for in situ drug delivery. Hence, the application of the local treatment may become a viable alternative to study intrafollicular factors [4,14,15] or a complement to COH. The current advances in nanotechnology and assisted reproductive tools are likely to boost the outcomes of intrafollicular interventions.

Numerous studies have used “naked” biomolecules, without a carrier, for intrafollicular injections [4,[16], [17], [18], [19]], which may limit their potential to gain mechanistic insights related to follicle growth and oocyte maturation. Interestingly, the recent detection of extracellular membrane microvesicles in equine [20], human [21], and bovine [22] follicular fluid (FF) and their roles as carriers for biomolecules’ delivery (e.g., lipids, proteins, RNAs, and miRNA) enabling intercellular communications have prompted new interest in the use of liposome nanoparticles for intrafollicular injections [[23], [24], [25]]. Liposomes are clinically relevant carbon-based nanoparticles shaped like spherical vesicles and constituted of phospholipid bilayers providing biocompatibility, biodegradability, and low cytotoxicity [26]. These nanoparticles have been used as effective carriers for controlled and rapid deliveries of hydrophilic and hydrophobic molecules to specific biological sites, with undeniable benefits during in vivo and in vitro applications [27,28]. Several drugs, including FF compounds [[29], [30], [31], [32], [33]], can be loaded into liposome vesicles for intrafollicular injections to achieve fertility treatments. Associated with intrafollicular injections, the minimally invasive in vivo Follicle Wall Biopsy (FWB) technique, a recently improved methodology for folliculogenesis studies, allows for simultaneous sampling of FF and follicle cells (granulosa, theca interna and externa cells). Most importantly, the FWB technique on living mares does not affect ovarian function [[34], [35], [36]], and the evolutive composition of the FF during antral follicle growth remains an excellent cocktail to search for biomarkers of clinical interests.

From this background, the current study aimed to develop a minimally invasive and efficient intrafollicular and intracellular drug delivery system, followed by a sampling of intrafollicular components to enable precise follicular health diagnostic through both molecular imaging and in-depth understanding of the mechanisms governing antral follicle growth and oocyte maturation. For those purposes, the effectiveness of an intrafollicular (or local) drug delivery system (liposome encapsulation) was evaluated through the application of intrafollicular (injection and flushing), FWB, and fluorescent imaging techniques permitting accurate assessment of the intracellular delivery. Therefore, fluorescent liposome nanoparticles were injected in vivo into ovarian antral follicles of living mares to visualize their binding to the plasma membrane of different follicular cells as the first step for future implementation of in situ fertility treatments and deep-tissue imaging. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential through the natural fluorescence of doxorubicin-DNA interaction.