*3.1. Liposomes Formulation and Characterization* **3. RESULTS**

Poly ethylene glycol (PEG)-stabilized and 5-FU-loaded LPs (5-FU/LPs) were decorated with anti-FZD10 and in vitro tested to evaluate their effectiveness for selective CRC treatment (Figure 1A). A schematic illustration of active targeting of CRC by molecular recognition between anti-FZD10/5-FU/LPs and FZD10, expressed at the plasma membrane surface of CRC cells, is shown in Figure 1B. *3.1. Liposomes Formulation and Characterization*  Poly ethylene glycol (PEG)-stabilized and 5-FU-loaded LPs (5-FU/LPs) were decorated with anti-FZD10 and in vitro tested to evaluate their effectiveness for selective CRC treatment (Figure 1A). A schematic illustration of active targeting of CRC by molecular recognition between anti-FZD10/5- FU/LPs and FZD10, expressed at the plasma membrane surface of CRC cells, is shown in Figure 1B.

**Figure 1.** Pictorial sketch of PEG (Poly ethylene glycol) stabilized and 5-Fluorouracil (5-FU)-loaded liposomes (LPs) before (5-FU/LPs) and after (anti-FZD10/5-FU/LPs) conjugation with FZD10 antibody, according to the corresponding legend (**A**). Schematic representation of molecular recognition between anti-FZD10/5-FU/LPs and FZD10 protein expressed on the colorectal cancer (CRC) cell membrane surface. Drawings not to scale. (**B**). **Figure 1.** Pictorial sketch of PEG (Poly ethylene glycol) stabilized and 5-Fluorouracil (5-FU)-loaded liposomes (LPs) before (5-FU/LPs) and after (anti-FZD10/5-FU/LPs) conjugation with FZD10 antibody, according to the corresponding legend (**A**). Schematic representation of molecular recognition between anti-FZD10/5-FU/LPs and FZD10 protein expressed on the colorectal cancer (CRC) cell membrane surface. Drawings not to scale. (**B**).

For the preparation of anti-FZD10/5-FU/LPs, 5-FU/LPs bearing carboxylic groups were covalently conjugated with the primary amine groups of anti-FZD10 antibody by crosslinking chemistry. The effective occurrence of the conjugation reaction between FZD10-antibody and 5- FU/LPs was assessed by labeling the surface of anti-FZD10/5-FU/LPs with a specific secondary antibody-Alexa Fluor 555. The indirect detection of FZD10-antibody on anti-FZD10/5-FU/LPs was demonstrated by UV-Vis absorbance spectroscopy analysis, as described in the Supplementary Materials. In particular, the presence of a peak centered at 555 nm, due to the covalent binding of the dye conjugated secondary antibody to the surface of anti-FZD10/5-FU/LPs, can be observed in the absorption spectrum of anti-FZD10/5-FU/LPs, after their incubation with labelled secondary antibody (Figure S1B, Supplementary Materials), while the same peak does not appear in the absorbance spectrum of untreated anti-FZD10/5-FU/LPs (Figure S1A, Supplementary Materials). For the preparation of anti-FZD10/5-FU/LPs, 5-FU/LPs bearing carboxylic groups were covalently conjugated with the primary amine groups of anti-FZD10 antibody by crosslinking chemistry. The effective occurrence of the conjugation reaction between FZD10-antibody and 5-FU/LPs was assessed by labeling the surface of anti-FZD10/5-FU/LPs with a specific secondary antibody-Alexa Fluor 555. The indirect detection of FZD10-antibody on anti-FZD10/5-FU/LPs was demonstrated by UV-Vis absorbance spectroscopy analysis, as described in the Supplementary Materials. In particular, the presence of a peak centered at 555 nm, due to the covalent binding of the dye conjugated secondary antibody to the surface of anti-FZD10/5-FU/LPs, can be observed in the absorption spectrum of anti-FZD10/5-FU/LPs, after their incubation with labelled secondary antibody (Figure S1B, Supplementary Materials), while the same peak does not appear in the absorbance spectrum of untreated anti-FZD10/5-FU/LPs (Figure S1A, Supplementary Materials).

The formulated LPs, namely 5-FU/LPs and anti-FZD10/5-FU/LPs, were characterized in terms of size, morphology and colloidal stability by performing DLS investigation, TEM analysis and ζpotential measurements (Table 1). DLS investigation revealed that the mean hydrodynamic diameter of bare 5-FU/LPs was equal to (155 ± 47) nm, and this domain size was a consequence of the porosity of the polycarbonate membrane used for the extrusion. Anti-FZD10/5-FU/LPs exhibited a larger The formulated LPs, namely 5-FU/LPs and anti-FZD10/5-FU/LPs, were characterized in terms of size, morphology and colloidal stability by performing DLS investigation, TEM analysis and ζ-potential measurements (Table 1). DLS investigation revealed that the mean hydrodynamic diameter of bare 5-FU/LPs was equal to (155 ± 47) nm, and this domain size was a consequence of the porosity of the polycarbonate membrane used for the extrusion. Anti-FZD10/5-FU/LPs exhibited a larger average size

average size (193 ± 12) nm, as expected when the surface functionalization of liposomes is carried out.

(Table 1).

remove the unbound antibody molecules.

(193 ± 12) nm, as expected when the surface functionalization of liposomes is carried out. The values of polydispersion index (PDI) denoted the presence of a fairly uniform LPs population in dimensional terms for both the formulations (Table 1).

**Table 1.** Intensity-average hydrodynamic diameter and corresponding polydispersity index (PDI) determined by Dynamic Light Scattering (DLS), ζ-potential, and encapsulation efficiency (EE%) of 5-FU/LPs and anti-FZD10/5-FU/LPs. **Table 1.** Intensity-average hydrodynamic diameter and corresponding polydispersity index (PDI) determined by Dynamic Light Scattering (DLS), ζ-potential, and encapsulation efficiency (EE%) of 5-

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The representative TEM micrographs of 5-FU/LPs (Figure 2A,A1) and FZD10-anti/5-FU/LPs (Figure 2B,B1) proved the formation of nanostructures with quite round shape for both the liposomal formulations. In the TEM micrographs of 5-FU/LPs (Figure 2A,A1) and anti-FZD10/5-FU/LPs (Figure 2B,B1), nanostructures with a rounded shape can be observed for both the formulated LPs, having size ranging from 30 to 110 nm and from 40 to 195 nm for 5-FU/LPs and FZD10-anti/5-FU/LPs, respectively. Furthermore, for both the bare and the engineered LPs, some nano-objects revealing a darker, circular bilayer and a brighter interior space, as typically ascribed to LP structures, can be appreciated, as highlighted in the two high magnification close-ups (Figure 2A1,B1). The representative TEM micrographs of 5-FU/LPs (Figure 2A,A1) and FZD10-anti/5-FU/LPs (Figure 2B,B1) proved the formation of nanostructures with quite round shape for both the liposomal formulations. In the TEM micrographs of 5-FU/LPs (Figure 2A,A1) and anti-FZD10/5-FU/LPs (Figure 2B,B1), nanostructures with a rounded shape can be observed for both the formulated LPs, having size ranging from 30 to 110 nm and from 40 to 195 nm for 5-FU/LPs and FZD10-anti/5-FU/LPs, respectively. Furthermore, for both the bare and the engineered LPs, some nano-objects revealing a darker, circular bilayer and a brighter interior space, as typically ascribed to LP structures, can be appreciated, as highlighted in the two high magnification close-ups (Figure 2A1,2B1).

**Figure 2.** Representative transmission electron microscopy (TEM) micrograph of 5-FU/LPs (**A**,**A1**) and anti-FZD10/5-FU/LPs (**B**,**B1**). High magnification close-up of 5-FU/LPs (**A1**) and anti-FZD10/5-FU/LPs (**B1**) along with the corresponding sketch. Scale bar 100 nm. **Figure 2.** Representative transmission electron microscopy (TEM) micrograph of 5-FU/LPs (**A**,**A1**) and anti-FZD10/5-FU/LPs (**B**,**B1**). High magnification close-up of 5-FU/LPs (**A1**) and anti-FZD10/5-FU/LPs (**B1**) along with the corresponding sketch. Scale bar 100 nm.

The TEM observations resulted in a good agreement with the data obtained by DLS investigation, also considering that the deposition procedure of samples on the TEM grid, performed before TEM analysis, induced a drying process and consequently a shrinking of the soft organic The TEM observations resulted in a good agreement with the data obtained by DLS investigation, also considering that the deposition procedure of samples on the TEM grid, performed before TEM analysis, induced a drying process and consequently a shrinking of the soft organic matter based LPs.

matter based LPs. The ζ-potential measurements highlighted the presence of an overall negative charge at the The ζ-potential measurements highlighted the presence of an overall negative charge at the surface of both investigated formulations, as the phosphate moieties of the phospholipids used

surface of both investigated formulations, as the phosphate moieties of the phospholipids used for

The decrease in drug EE% recorded for anti-FZD10/5-FU/LPs can be reasonably explained by taking into account the different steps required for the conjugation reaction, starting from bare 5- FU/LPs: the first incubation with the crosslinking agents, the subsequent centrifugation to remove the excess of reagents, the second incubation with the FZD10 antibody and the final centrifugation to for the preparation of 5-FU/LPs and anti-FZD10/5-FU/LPs are expected to be exposed onto their surface (Table 1).

The decrease in drug EE% recorded for anti-FZD10/5-FU/LPs can be reasonably explained by taking into account the different steps required for the conjugation reaction, starting from bare 5-FU/LPs: the first incubation with the crosslinking agents, the subsequent centrifugation to remove the excess of reagents, the second incubation with the FZD10 antibody and the final centrifugation to remove the unbound antibody molecules.

The drug release study for FZD10-anti/5-FU/LPs, monitored by UV-Vis absorption spectroscopy, is reported in Figure S2 of Supplementary Materials.
