*2.4. Animal Studies, In Vivo Model*

In vivo experiments were designed and performed to minimize the use of animals. C57BL/6 mice (12–16 weeks old) were housed with a 12 h light/dark cycle with free provision of food and water at the Experimentation Service (SEEA) of the University of Cantabria. Animals were maintained, handled, and sacrificed following the directive 2010/63/UE. The B16-F10 lung metastasis model in the C57BL/6 strain of mice is well established in the literature [31–34]. This cell line was derived from a spontaneous melanoma developed in C57BL/6 mice [31]. Metastatic foci were produced upon intravenous B16-F10

malignant melanoma cell transplantation. For this purpose, ca. 100,000 B16-F10 melanoma cells (50 µL/mouse) were injected in the retro-orbital venous sinus using a 0.3 mL microsyringe (BD Micro-FineTM; USA). Ten days after intravenous cancer cell injection, mice were randomly divided into four groups: untreated controls, mice injected with free DOX, mice injected with control SLPs, and finally, mice injected with SLPs-DOX. Animals were treated 3 times every 2 days at a concentration of 2.5 mg/kg DOX (each doses). Mice were euthanized 20 days after the transplant and had their tissues collected and fixed in formalin for histology. Lung metastatic colonies were easily recognized as black spots on the lung surface [35]. Histopathological evaluation was performed on hematoxylin (1.09249.0500; Merck; Kenilworth, NJ, USA) and eosin (256879; Panreac; Barcelona, Spain) stained paraffinembedded lung tissue sections. Graphics were designed by BioRender.com.
