*3.4. E*ff*ectiveness of the Formulated Liposomes on Cell Migration*

The effect of the two formulated LPs on cell motility was assessed by performing the scratch assay on CoLo-205 and CaCo-2 cells. After creating the mechanical scratch (marked in red) on confluent cell monolayers, the cells were incubated with exogenous added free 5-FU, 5-FU/LPs or anti-FZD10 /5-FU/LPs (drug concentration 2 µM) and the effects were monitored at 0, 24 and 48 h (Figure 6A,B). CoLo-205 cells were able to wholly rescue the wound already within 24 h (Figure 6A), while for the nonmetastatic, untreated CaCo-2 cells, complete closure of the scratched area was observed within 48 h (Figure 6B). In the case of CoLo-205 cells, free 5-FU or 5-FU/LPs decreased cell migration after 24 h, although the widths of the injuries were still detectable. At 48 h, the scratched area appeared strongly reduced. Conversely, incubation with anti-FZD10/5-FU/LPs produced a substantial inhibition of cell migration in the wound regions; indeed, the scratched area was only partially reduced but still observable at both 24 and 48 h (Figure 6A). The results obtained by the scratch assay performed on CaCo-2 cells demonstrated a notable inhibition effect of 5-FU, free or encapsulated in the LPs, on cell migration (Figure 6B). Indeed, a considerable decrease of cell migration was observed, as compared with control cells, when the cells were exposed to the free 5-FU, although the area of injury appeared partially reduced at 24 and 48 h. In the case of 5-FU/LPs, CaCo-2 cells were able to partially migrate in the scratched area at 24 h, but the injury region was no longer recognizable at 48 h, since the cells appeared to be dead. The effect of anti-FZD10/5-FU/LPs on the migration and viability of CaCo-2 cells was more pronounced than that of nontargeted LPs; indeed, cell migration did not occur and death of the cells was already observed at 24 h. Remarkably, almost all the cells appeared to be dead at 48 h (Figure 6B).

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**Figure 6.** Qualitative analysis of collective CoLo-205 (**A**) and CaCo-2 (**B**) cell migration by in vitro scratch assay. Representative photomicrographs of scratch-wound closure of cells treated with free 5- FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at different time points (0, 24 and 48 h). 5-FU concentration: 2 µM. CTR: untreated cells. Red lines represent the edges of the scratched areas. Scale bar: 200 µm. **Figure 6.** Qualitative analysis of collective CoLo-205 (**A**) and CaCo-2 (**B**) cell migration by in vitro scratch assay. Representative photomicrographs of scratch-wound closure of cells treated with free 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at different time points (0, 24 and 48 h). 5-FU concentration: 2 µM. CTR: untreated cells. Red lines represent the edges of the scratched areas. Scale bar: 200 µm.

#### *3.5. Effectiveness of Formulated LPs on Vimentin and Phospho-paxillin Cytoskeletal Proteins 3.5. E*ff*ectiveness of Formulated LPs on Vimentin and Phospho-Paxillin Cytoskeletal Proteins*

The expression level of two specific proteins, namely vimentin and phospho-paxillin, involved in cell stability and cell adhesion, respectively, was assessed by immunofluorescence imaging in both fixed CoLo-204 and CaCo-2 cells, after incubation with 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at 24 and 48 h. The tested 5-FU concentration was 2 µM (Figure 7). The confocal microscopy investigation performed on CoLo-205 cells treated with free 5-FU (Figure 7A–C) indicated a significant, timedependent reduction of vimentin expression (green labeled), being (36.8 ± 7.5)% and (48.6 ± 11.2)% the percentage ratio (compared to the control) of mean fluorescence intensity for the cells treated at 24 and 48 h, respectively (p < 0.001 versus control). Conversely, in the case of phosphor-paxillin (red labeled), the percentage ratio of fluorescence intensity was firstly increased, at 24 h ((139.1 ± 19.3)% and then reduced to (69.3 ± 7.4)% at 48 h (p < 0.001 versus control). The expression level of two specific proteins, namely vimentin and phospho-paxillin, involved in cell stability and cell adhesion, respectively, was assessed by immunofluorescence imaging in both fixed CoLo-204 and CaCo-2 cells, after incubation with 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at 24 and 48 h. The tested 5-FU concentration was 2 µM (Figure 7). The confocal microscopy investigation performed on CoLo-205 cells treated with free 5-FU (Figure 7A–C) indicated a significant, time-dependent reduction of vimentin expression (green labeled), being (36.8 ± 7.5)% and (48.6 ± 11.2)% the percentage ratio (compared to the control) of mean fluorescence intensity for the cells treated at 24 and 48 h, respectively (*p* < 0.001 versus control). Conversely, in the case of phosphor-paxillin (red labeled), the percentage ratio of fluorescence intensity was firstly increased, at 24 h ((139.1 ± 19.3)% and then reduced to (69.3 ± 7.4)% at 48 h (*p* < 0.001 versus control).

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**Figure 7.** Detection and quantification of vimentin and phospho-paxillin (red) by immunofluorescence confocal microscopy in fixed CoLo-205 (**A**–**C**) and CaCo-2 cells (**D**–**F**), after cells incubation with free 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at 24 and 48 h. 5-FU concentration: 2 µM. CTR: untreated cells. Green channel: labeled vimentin, red channel: labeled phospho-paxillin, blue channel: labeled nuclei (DAPI), and corresponding overlay (Merge). Scale bar: 50 µm. (\*) p < **Figure 7.** Detection and quantification of vimentin and phospho-paxillin (red) by immunofluorescence confocal microscopy in fixed CoLo-205 (**A**–**C**) and CaCo-2 cells (**D**–**F**), after cells incubation with free 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at 24 and 48 h. 5-FU concentration: 2 µM. CTR: untreated cells. Green channel: labeled vimentin, red channel: labeled phospho-paxillin, blue channel: labeled nuclei (DAPI), and corresponding overlay (Merge). Scale bar: 50 µm. (\*) *p* < 0.001 versus control (CRT).

0.001 versus control (CRT). The confocal images of CoLo-205 cells after treatment with 5-FU/LPs or anti-FZD10/5-FU/LPs show that a significant reduction (p < 0.001 versus control) of the expression level of each investigated protein was always observed at both cell incubation times. Indeed, in the case of vimentin, the percentage ratios of mean fluorescence intensity were equal to (38.4 ± 6.3)% and (26.9 ± 4.9)% at 24 h and (19.3 ± 2.4)% and (12.8 ± 5.5)% at 48 h, for cells treated with 5-FU/LPs or anti-FZD10/5-FU/LPs, respectively; in the case of phospho-paxillin, the corresponding percentage ratios were (53.9 ± 12.7)% and (55.0 ± 5.5)% at 24 h and (49.9 ± 3.4)% and (9.7 ± 2.5)% at 48 h (Figure 7A–C). The immunofluorescence analysis carried out by confocal microscopy on CaCo-2 cells treated with free 5-FU (Figure 7D–F) demonstrated that although the expression levels of vimentin and phosphopaxillin decreased in a time-dependent manner, the observed decreases of fluorescent signals were not significant. On the contrary, a significant decrease (p < 0.001 versus control) in the expression levels of vimentin and phospho-paxillin was observed when CaCo-2 cells were incubated with 5- FU/LPs for 48 h. The percentage ratio of mean fluorescence intensity was found to be (28.2 ± 15.2)% and (14.0 ± 3.5)% for vimentin and phospho-paxillin, respectively (Figure 7D,E,F). A significant (p < 0.001 versus control), detectable decrease in the expression level of phospho-paxillin was observed in CaCo-2 cells treated with anti-FZD10/5-FU/LPs for 24 and 48 h (percentage ratio of mean fluorescence intensity (21.7 ± 0.5)% and (5.3 ± 0.7)% at 24 and 48 h, respectively), while in the case of vimentin, a significant difference in the expression level, compared to the control (p < 0.001 versus control), was recorded only at 48 h (percentage ratio of mean fluorescence intensity (5.2 ± 4.6)%, (Figure 7D,E,F)). For both cell lines, the reductions in fluorescence intensity observed for the bluestained cell nuclei, compared to the corresponding control cells, when the cells were treated with the The confocal images of CoLo-205 cells after treatment with 5-FU/LPs or anti-FZD10/5-FU/LPs show that a significant reduction (*p* < 0.001 versus control) of the expression level of each investigated protein was always observed at both cell incubation times. Indeed, in the case of vimentin, the percentage ratios of mean fluorescence intensity were equal to (38.4 ± 6.3)% and (26.9 ± 4.9)% at 24 h and (19.3 ± 2.4)% and (12.8 ± 5.5)% at 48 h, for cells treated with 5-FU/LPs or anti-FZD10/5-FU/LPs, respectively; in the case of phospho-paxillin, the corresponding percentage ratios were (53.9 ± 12.7)% and (55.0 ± 5.5)% at 24 h and (49.9 ± 3.4)% and (9.7 ± 2.5)% at 48 h (Figure 7A–C). The immunofluorescence analysis carried out by confocal microscopy on CaCo-2 cells treated with free 5-FU (Figure 7D–F) demonstrated that although the expression levels of vimentin and phospho-paxillin decreased in a time-dependent manner, the observed decreases of fluorescent signals were not significant. On the contrary, a significant decrease (*p* < 0.001 versus control) in the expression levels of vimentin and phospho-paxillin was observed when CaCo-2 cells were incubated with 5-FU/LPs for 48 h. The percentage ratio of mean fluorescence intensity was found to be (28.2 ± 15.2)% and (14.0 ± 3.5)% for vimentin and phospho-paxillin, respectively (Figure 7D–F). A significant (*p* < 0.001 versus control), detectable decrease in the expression level of phospho-paxillin was observed in CaCo-2 cells treated with anti-FZD10/5-FU/LPs for 24 and 48 h (percentage ratio of mean fluorescence intensity (21.7 ± 0.5)% and (5.3 ± 0.7)% at 24 and 48 h, respectively), while in the case of vimentin, a significant difference in the expression level, compared to the control (*p* < 0.001 versus control), was recorded only at 48 h (percentage ratio of mean fluorescence intensity (5.2 ± 4.6)%, (Figure 7D–F)). For both cell lines, the reductions in fluorescence intensity observed for the blue-stained cell nuclei, compared to the corresponding control cells, when the cells were treated with the two formulated LPs or free 5-FU at 24 and 48 h, were in agreement with the results obtained at the cell viability assay (Figure 3).
