Validation of lipo-LOX activity

To confirm the activity of LOX antibody after the liposome functionalization, secreted LOX protein was quantified in the cells culture medium at different time points. Briefly, MDA-MB-231 cultured both in standard monolayer and tridimensional culture were exposed to Lipo, EPI, Lipo-EPI, and Lipo-EPI-LOX at the concentration of the human plasma peak as specified in the drug testing section. Culture medium was collected at 6, 24, and 48 h respectively. The medium was centrifuged at 15,000 rpm for 20 min at 4 °C and the pellet was discarded to eliminate cell debris. The protein contents were determined using a BCA protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific). An equal amount of protein from each sample was separated on Criterion Precast Gel Tris–HCl (Biorad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore Corporation)⁴⁴. The membranes were blocked for 2 h with a solution containing 5% fat-free milk PBS with 0.1% Tween 20 (Sigma-Aldrich) at room temperature and incubated overnight at 4 °C with anti-LOX antibody (1:1000 ab31238, Abcam, Cambridge, UK). The membranes were then washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody following the protocol previously reported⁴⁵.