*3.2. E*ff*ectiveness of Formulated Liposomes on Cell Viability*

CoLo-205 and CaCo-2 cells were selected as FZD10-positive CRC cell lines (Figure S3, Supplementary Materials). The effect of 5-FU and anti-FZD10/LPs on CoLo-205 and CaCo-2 cells viability was evaluated by MTS proliferation assay. For comparison, free 5-FU and nontargeted LPs were tested. The effect of the empty LPs (having a ζ-potential value of (−32.84 ± 3.42) mV) on cell viability was also assessed, in order to investigate on the cytotoxicity of liposomal vectors. In particular, the two cell lines were treated with free 5-FU, 5-FU/LPs and anti-FZD10/5-FU/LPs at drug concentrations ranging from 1 to 10 µM (in the range from 5 to 50 µM, in terms of total lipid concentration for the liposomal formulations), at 24, 48, and 96 h. CoLo-205 and CaCo-2 cell viability is reported in Section 3.3. The viability of both colon cancer cell lines was always greater than 80% after their incubation with empty LPs, thus indicating very low toxicity in the lipid concentration range, at each tested time incubation (Figure 3, yellow bars). For CoLo-205 cells, at the lowest tested drug concentration values, namely 1 and 2 µM, the cell viability was only minimally reduced (always higher than 75%) within 96 h, when cells were incubated with free 5-FU (Figure 3A, blue bars). As compared to free 5-FU, the nontargeted LPs were found to exhibit an enhanced cytotoxic activity, for each tested time incubation, at a drug concentration of 2 µM (Figure 3, grey bars). Conversely, in the range between 3 and 10 µM, the effectiveness of free 5-FU in affecting the cell viability was time-dependent (Figure 3A, blue bars); the bare 5-FU/LPs induced a higher cytotoxic effect than free 5-FU by reducing the cell viability up to about 40% only within the first 24 h (Figure 3A, grey bars). In the case of anti-FZD10/5-FU/LPs, the cell-killing effects were always time-dependent from 24 to 96 h, for each tested drug concentration (Figure 3A, orange bars). The cell viability was always lower than 30% when the cells were treated with anti-FZD10/5-FU/LPs for 96 h, in the entire tested range of drug concentrations (Figure 3A, orange bars). At a 5-FU concentration of 2 µM, the use of anti-FZD10/5-FU/LPs significantly (*p* < 0.001 versus control) reduced the cell viability up to (42 ± 3) and (28 ± 8)% at 48 and 96 h, while in the case of free 5-FU, only up to (74 ± 4) and (73 ± 10)%, thus resulting not statistically significant (Figure 3A, orange and blue bars). The cell viability recorded for nontargeted 5-FU/LPs was equal to (55.9 ± 6.6) and (42.9 ± 2.3)% when cells were treated at drug concentration of 2 µM for 48 and 96 h, respectively, thus highlighting their less significant cytotoxic efficacy respect to targeted anti-FZD10/5-FU/LPs.

For CaCo-2 cells, the bare 5-FU/LPs exhibited a time-dependent reduction of cell viability ranging from approximately 60% to 40% over the 24 to 96 h, for all tested concentrations (Figure 3B, grey bars). When the cells were incubated with free drug at the lowest tested concentrations (1 and 2 µM), the cell viability was only minimally affected at 24 and 48 h; conversely, a substantial reduction (*p* < 0.001 versus control) up to (38 ± 8)% in cell viability was recorded at 96 h (Figure 3B, blue bars). At the higher explored drug concentrations (3–10 µM), the free 5-FU exhibited a significant cytotoxic activity (*p* < 0.001 versus control) not only at 96, but also at 48 h (Figure 3B, blue bars). Cells exposure to anti-FZD10/5-FU/LPs induced a relevant cell-killing effect already within 24 h, throughout the drug concentrations range (Figure 3B, orange bars). Cell treatment with the engineered LPs significantly lowered (*p* < 0.001 versus control) the cell viability up to (40 ± 4) and (37 ± 6)% at 24 h, and up to (13 ± 2) and (16 ± 8)% at 96 h, at the tested drug concentrations equal to 1 and 2 µM, respectively. (Figure 3B, orange bars).

*Pharmaceutics* **2020**, *12*, x 9 of 19

**Figure 3.** Cell viability, evaluated by MTS cell proliferation assay, of CoLo-205 (**A**) and CaCo-2 (**B**) cells after incubation with free 5-FU, 5-FU/LPs, anti-FZD10/5-FU/LPs and empty LPs at 5-FU concentrations ranging from 1 to 10 µM (from 5 to 50 µM in terms of total lipid concentration) for 24, 48 and 96 h. For each cell line, control was untreated cells. The experiments were conducted in triplicate. (\*) p < 0.001 versus control. **Figure 3.** Cell viability, evaluated by MTS cell proliferation assay, of CoLo-205 (**A**) and CaCo-2 (**B**) cells after incubation with free 5-FU, 5-FU/LPs, anti-FZD10/5-FU/LPs and empty LPs at 5-FU concentrations ranging from 1 to 10 µM (from 5 to 50 µM in terms of total lipid concentration) for 24, 48 and 96 h. For each cell line, control was untreated cells. The experiments were conducted in triplicate. (\*) *p* < 0.001 versus control.

#### For CaCo-2 cells, the bare 5-FU/LPs exhibited a time-dependent reduction of cell viability *3.3. E*ff*ects of Formulated Liposomes on the Cell Morphology*

*3.3. Effects of Formulated Liposomes on the Cell Morphology* 

respectively. (Figure 3B, orange bars).

ranging from approximately 60% to 40% over the 24 to 96 h, for all tested concentrations (Figure 3B, grey bars). When the cells were incubated with free drug at the lowest tested concentrations (1 and 2 Morphological changes induced on CoLo-205 and CaCo-2 cells by exposure to anti-FZD10/5-FU/LPs and 5-FU/LPs (drug concentration of 2 µM) at 6, 24 and 96 h were observed at FE-SEM (Figures 4 and 5).

µM), the cell viability was only minimally affected at 24 and 48 h; conversely, a substantial reduction (p < 0.001 versus control) up to (38 ± 8)% in cell viability was recorded at 96 h (Figure 3B, blue bars). At the higher explored drug concentrations (3–10 µM), the free 5-FU exhibited a significant cytotoxic activity (p < 0.001 versus control) not only at 96, but also at 48 h (Figure 3B, blue bars). Cells exposure to anti-FZD10/5-FU/LPs induced a relevant cell-killing effect already within 24 h, throughout the drug concentrations range (Figure 3B, orange bars). Cell treatment with the engineered LPs significantly lowered (p < 0.001 versus control) the cell viability up to (40 ± 4) and (37 ± 6)% at 24 h, and up to (13 ± 2) and (16 ± 8)% at 96 h, at the tested drug concentrations equal to 1 and 2 µM, In Figure S4 (Supplementary Materials), the pristine morphology of the untreated CaCo-2 and CoLo-205 cells was shown. The two cell lines appeared adherent, CaCo-2 cells are completely flat, while the CoLo-205 cells present a protruding spherical nucleus in the center of the flat cell body (Figure S4). FE-SEM analysis performed on both cell lines after treatment with 5-FU/LPs and anti-FZD10/5-FU/LPs for 6 h revealed that, the two liposomal formulations induced rounding of the cells, that acquired a spindle-shaped morphology as compared to the corresponding controls, with an increased number of pseudopodia due to the stress induced by incubation with 5-FU/LPs or anti-FZD10/5-FU/LPs (Figure 4A,A1,B,B1, and Figure 5A,A1,B,B1).

(Figures 4 and 5).

Morphological changes induced on CoLo-205 and CaCo-2 cells by exposure to anti-FZD10/5-

**Figure 4.** (FE-SEM) Representative field emission scanning electron microscopy micrographs(EHT) extra-high tension (EHT = 3.00 kV) and their corresponding close-up details at higher magnification of CoLo-205 cells after treatment with 5-FU/LPs (**A**,**A1**,**C**,**C1**,**E**,**E1**) and anti-FZD10/5-FU/LPs (**B**,**B1**,**D**,**D1**,**F**,**F1**) for 6, 24 and 96 h. 5-FU concentration: 2 µM. **Figure 4.** (FE-SEM) Representative field emission scanning electron microscopy micrographs(EHT) extra-high tension (EHT = 3.00 kV) and their corresponding close-up details at higher magnification of CoLo-205 cells after treatment with 5-FU/LPs (**A**,**A1**,**C**,**C1**,**E**,**E1**) and anti-FZD10/5-FU/LPs *Pharmaceutics*  (**B**,**B1**,**D**,**D1**,**F**,**F1**) for 6, 24 and 96 h. 5-FU concentration: 2 µM. **2020**, *12*, x 11 of 19

**Figure 5.** Representative FE-SEM micrographs (EHT = 3.00 kV) and their corresponding close-up details at higher magnification of CaCo-2 cells after treatment with 5-FU/LPs (**A**,**A1**,**C**,**C1**,**E**,**E1**) and anti-FZD10/5-FU/LPs (**B**,**B1**,**D**,**D1**,**F**,**F1**) for 6, 24 and 96 h. 5-FU concentration: 2 µM. **Figure 5.** Representative FE-SEM micrographs (EHT = 3.00 kV) and their corresponding close-up details at higher magnification of CaCo-2 cells after treatment with 5-FU/LPs (**A**,**A1**,**C**,**C1**,**E**,**E1**) and anti-FZD10/5-FU/LPs (**B**,**B1**,**D**,**D1**,**F**,**F1**) for 6, 24 and 96 h. 5-FU concentration: 2 µM.

After 96 h exposure to both the liposomal formulations, CoLo-205 cells appear dead (Figure 4E,E1 and F,F1), since no living cell structures were observed. The effectiveness of anti-FZD10/5- In the case of CoLo-205 cells, the 5-FU/LPs produced a more evident toxic effect compared to the anti-FZD10/5-FU/LPs after 24 h of incubation, revealing significant alterations in cell morphology and

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 and 6B). 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,

for 96 h (Figure 5E,E1 and F,F1).

*3.4. Effectiveness of the Formulated Liposomes on Cell Migration* 

almost all the cells appeared to be dead at 48 h (Figure 6B).

a strongly reduced number of pseudopodia (Figure 4C,C1,D,D1). Conversely, in the case of CaCo-2 cells, the anti-FZD10/5-FU/LPs mostly affected the cell morphology as compared to 5-FU/LPs. Indeed, CaCo-2 cell still resulted adherent to the substrate after treatment with the nontargeted liposomal formulations for 24 h (Figure 5C,C1,D,D1).

After 96 h exposure to both the liposomal formulations, CoLo-205 cells appear dead (Figure 4E,E1,F,F1), since no living cell structures were observed. The effectiveness of anti-FZD10/5-FU/LPs was also detectable on CaCo-2 cells at 96 h, since only cell fragments can be observed; conversely, a kind of morphology was still detected when CaCo-2 cells were treated with 5-FU/LPs for 96 h (Figure 5E,E1,F,F1).
