*3.4. Inhibition of Metastasis Growth or Antimetastatic Efficacy In Vivo by SLPs-DOX*

To investigate the ability of SLPs-DOX to inhibit melanoma metastasis in the lungs, we used a well-established mouse metastatic model using B16-F10 melanoma cells as described in Section 2.4. For the study, metastasis-bearing animals were treated intravenously with 3 doses of 2.5 mg/kg of free DOX, or the equivalent amount of encapsulated drug in SLPs-DOX, at 10, 12, and 14 days after metastasis induction (Figure 3). Animals were sacrificed 20 days after the initial cell transplant. Lungs were collected, photographed, and preserved by fixation in formalin. After 20 days of intravenous injection of the cells, black colonies of metastatic cells were clearly observable on the surface of the lungs of all injected mice (Figure 4, negative control).

**Figure 3.** Schematic representation of the metastasis growth inhibition experiment.

**Figure 4.** Representative images of metastatic lungs for each experimental group. In the first row, metastatic foci are recognized as black spots on the fresh lung surface. Hematoxylin–eosin-stained histological sections of the lung tissues are shown. Metastatic tissues are indicated by asterisks.

Figure 4 shows a representative example of the lungs after the different treatments. DOX-treated lungs (whether free or SLPs-DOX) present less metastatic spots than negative controls. In fact, SLPs-DOX-treated lungs present the lowest amount of metastasis. Histological analysis corroborates the results from fresh lung images.

ImageJ software was used to quantify the affected area in the lungs to evaluate the effect of treatment on metastasis. The ratio of affected area/total lung area was calculated in 120 animals. Figure 4 shows representative images of the lungs of mice treated with SLPs-DOX, where approximately a 60% reduction of the affected area was observed compared with mice treated with free DOX. These data, together with the differences between the SLPs-DOX-treated mice and the untreated mice, are statistically significant (Figure 5). It is also important to note that no significant differences were found in the survival rates (Figure S5) or body weight (Figure S6) between mice from the different treatment groups, indicating no detectable SLPs-DOX in vivo toxicity during the timeframe of these tests.

**Figure 5.** SLPs significantly reduce metastasis in the lungs. Representation of lung metastasis is expressed as the affected area with respect to the total area of the lung. Reduction in metastasis can be observed when the lungs are treated with the SLPs-DOX compared with the lungs treated with the free drug (*t* = 5.94; *n* = 170; \* t0.001). These differences are also significant between the lungs treated with SLPs-DOX with respect to untreated lungs.
