*2.2. Evaluation of Encapsulation E*ffi*ciency (EE%)*

The encapsulation efficiency (EE%) of 5-FU encapsulated in 5-FU/LPs or anti-FZD10/5-FU/LPs was evaluated according to the following formula:

$$\text{EE\%} = \text{Wt} \text{\%} \text{Wi} \times 100 \tag{1}$$

where Wt is the amount of drug effectively incorporated into the liposomal formulation and Wi the total quantity of 5-FU initially added during the preparation. To evaluate the drug content, samples were lyophilized and treated with methanol (1:100 dilution) and then the absorbance spectra (Perkin Elmer Double beam UV-Visible Spectrophotometer Lambda Bio 20) were recorded at 265 nm versus a methanol solution containing the same lipid mixture used for liposome preparation (baseline) [20]. Three measurements were performed on three different batches for each liposomes formulation. A calibration curve of 5-FU was generated by measuring the drug absorbance at 265 nm of standard methanol solutions at concentrations ranging from 0.25 mM to 0.05 mM.
