Liposomes (LPs) are nanoscale vesicles composed of one or more phospholipid bilayers that self-assemble in aqueous environments. Their amphiphilic structure enables the simultaneous incorporation of hydrophilic compounds in their aqueous core and hydrophobic compounds within the lipid bilayer, making them highly versatile nanocarriers. Owing to their biocompatibility, structural similarity to cell membranes, and modifiable surface properties, LPs have been extensively studied for the delivery of bioactive compounds to specific cells and tissues. Since their discovery in the 1960s, they have found wide application across pharmaceuticals, cosmetics, food, agriculture, and biomedical imaging. In particular, LPs have shown great promise in drug delivery systems by enhancing the solubility, stability, and targeted release of bioactive molecules while minimizing off-target effects [1]. Moreover, in biomedical and reproductive sciences, optimizing LPs formulations to improve cellular uptake and modulate molecular pathways has emerged as a powerful strategy for both therapeutic interventions and experimental studies [2], [3], [4].

The physicochemical properties of liposomes can be extensively tailored through the selection of specific lipid components. The available lipids, obtained from both natural and synthetic origins, differ in head group charge, acyl chain length, and saturation [5]; [6]. These characteristics directly influence liposome surface charge, fluidity, and interaction with biological membranes. Cationic lipids confer a net positive charge, facilitating electrostatic interactions with negatively charged biomolecules such as DNA and RNA, and with cellular membranes [7]. On the other hand, anionic lipids, which carry a negative charge, more closely resemble natural membrane systems [8]. However, due to the high curvature typical of nanoscale vesicles, achieving proper lipid packing and bilayer stability can be challenging. Cholesterol is commonly incorporated into liposome membranes to address this issue, as it integrates into the hydrophobic acyl chain region to fill the gaps between phospholipids, enhancing membrane rigidity and structural stability [9]. Additionally, cholesterol helps to reduce membrane permeability and contributes to increasing the stability of LPs in vivo and to reducing the exchange of lipid molecules between LPs and circulating cells [10], [11], [12].

Despite the promising potential of LPs, traditional delivery systems targeting specific cell types often face limitations, including low cellular specificity and poor bioavailability. These challenges are particularly relevant in reproductive biology, where the precise delivery of regulatory molecules to ovarian cells could significantly improve outcomes related to fertility and follicular development (Józkowiak et al., 2023; Obedkova et al., 2025). Nanocarriers such as LPs offer a compelling alternative, with their tunable surface properties and ability to encapsulate diverse molecular payloads, positioning them as effective tools for targeted interventions.

Granulosa cells (GCs), essential components of the ovarian follicle, play a critical role in folliculogenesis, steroid hormone production, and oocyte maturation [13], [14]. Their metabolic and functional activity is tightly regulated by intra- and extracellular signaling mechanisms, including those involving lipid metabolism and apoptosis [15], [16]. Understanding and manipulating these pathways can provide valuable insights into ovarian physiology and fertility regulation. In this context, LPs could offer an interesting platform to deliver regulatory molecules or nutrients directly to GCs; however, the efficacy of LP-based delivery systems depends on their physicochemical characteristics, including size, surface charge, and stability, which influence their interaction with cells. Specifically, the surface charge, whether anionic or cationic, can significantly affect cellular uptake, cytotoxicity, and intracellular fate [17], [18]. Although cationic LPs (CatLPs) have been widely studied for gene delivery applications, no studies have systematically compared their biological effects with anionic LPs (AnLPs) in GCs, particularly in terms of uptake efficiency and gene modulation. Granulosa cells are an established model for investigating ovarian function, given their physiological similarities among species and their relevance in both fertility research and livestock management [19]. Additionally, improving the health and functionality of these cells through nanocarrier-based supplementation may reduce the reliance on repeated hormonal treatments in assisted reproduction, offering a more sustainable and animal-friendly strategy in agricultural settings [20].

In this study, we produced and characterized both anionic and cationic liposomal formulations by tip sonication using hydrogenated and nonhydrogenated phospholipids and subsequently evaluated their interaction with bovine GCs. We then evaluated how these formulations interact with bovine GCs, examining cell viability, uptake efficiency, and gene expression responses over a continuous 12 h supplementation without medium replacement. Gene expression analysis focused on pathways related to steroidogenesis, apoptosis, and lipid metabolism, providing insights into the short-term cellular impact of liposome exposure under stringent conditions. While this study did not aim to optimize genetic material delivery, it established a new framework demonstrating the feasibility and biological relevance of these liposomal nanocarriers in reproductive biology. By integrating physicochemical characterization with functional cellular evaluation, our work provides a strong foundation for future studies exploring liposome-mediated delivery strategies in animal biotechnology.