*2.1. Preparation of Liposomes*

Nude LPs loaded with 5-FU (5-FU/LPs) were prepared using the ether injection method [24]. Briefly, 2.738 mL of an ether/chloroform (1:1, *v*/*v*) solution, 0.274 mL of a chloroform solution of lipid mix containing cholesterol, phosphatidylcholine and stearylamine (7:3:1 molar ratio) and 11 µL of a chloroform solution of DSPE-PEG2000 were mixed in a glass vial using a magnetic stirrer, brought to a lipid mix/DSPE-PEG2000 molar ratio of 3:0.3. Then, 3 mL of aqueous solution 5-FU (38 mM) were rapidly injected into the organic lipid solution using a sterile glass syringe. LPs were then obtained according to the previously reported experimental procedure [24]. Empty LPs were prepared following the same protocol, but without 5-FU addition.

For the preparation of 5-FU/LPs conjugated with FZD10 antibody (anti-FZD10/5-FU/LPs), 5-FU/LPs were previously surface-functionalized with carboxylic groups (5-FU/LPs-COOH). For this purpose, the above-described experimental protocol was followed, using as starting lipid mixture, 2.738 mL of an ether/chloroform (1:1, *v*/*v*) solution, 0.274 mL of a chloroform solution of lipid mix, 3 µL of a chloroform solution of DSPE-PEG2000 and 8 µL of a chloroform solution of DSPE-PEG2000-COOH. The final total lipid concentration in all the liposomal formulations was kept constant at 3 mM. After the purification procedure, 5-FU/LPs-COOH (300 µL) dispersed in ultrapure distilled water were activated by adding sulfo-NHS (11 mg) and EDC (9 mg). The reaction mixture was left under gentle stirring overnight at room temperature. Then, the samples were ultracentrifuged at 10,000× *g* for 40 min at 4 ◦C to remove excess crosslinking reagents. The activated 5-FU/LPs-COOH were recovered as pellets, dispersed in 300 µL of PBS and incubated with 5 µg of anti-FZD10 antibody. The mixture was gently stirred overnight at room temperature. Finally, the anti-FZD10/5-FU/LPs were purified by ultracentrifugation at 10,000× *g* at 4 ◦C for 40 min to remove unbound antibody. All the liposomal formulations were lyophilized (Christal freeze dryer alpha 1-4 LSC) and then reconstituted in PBS or water prior to their use or characterization. The experimental details on the indirect detection of FZD10-antibody bound onto the surface of LPs are reported in Supplementary Materials.
