Liposomal Encapsulation Reduces the Cytotoxic Effects of Gramicidin S in Monolayer and Spheroid Fibroblast Cultures

<b>Background/Objectives:</b> Gramicidin S (GS) is a cyclic antimicrobial peptide with strong antibacterial activity but significant cytotoxicity toward mammalian cells. This study evaluated GS-induced cytotoxicity in L929 fibroblast cells using both traditional 2D monolayer cultures and more physiologically relevant 3D spheroid models, and assessed whether liposomal encapsulation could mitigate toxicity and improve biocompatibility. <b>Methods:</b> L929 cells were cultured in monolayers and spheroids and treated with free GS or GS encapsulated in liposomes of varying lipid compositions. Cell viability and morphology were evaluated after 24 h of exposure using standard cytotoxicity assays. <b>Results:</b> Control liposomes, regardless of tested lipid type or concentration, showed no adverse effects on cell morphology or viability. Free GS caused pronounced, dose-dependent cytotoxicity in monolayers, decreasing viability to 11.0 ± 1.9% and 0.5 ± 1.1% at 50 and 75 µg/mL, respectively. By contrast, encapsulation in liposomes significantly reduced toxicity (<i>p</i> < 0.05), preserving 80.3-82.2% viability at 75 µg/mL depending on formulation, corresponding to protection factors exceeding 160-fold (80.3% vs. 0.5%). Spheroid cultures showed slightly higher resistance to GS; free GS reduced viability to 2.9%, while liposomal GS preserved it above 84.8%, depending on lipid composition. <b>Conclusions:</b> Liposomal encapsulation effectively reduces GS-induced cytotoxicity, likely by limiting direct membrane disruption. Moreover, spheroid models provide a more physiologically relevant and predictive platform for toxicity testing, while the results support nanoliposomes as a practical delivery strategy to enhance the safety of antimicrobial peptides during preclinical development.