Cytotoxicity assay

A cytotoxicity assay of the blank RGD-KLA-Lips was conducted using NIH 3T3 fibroblasts. NIH 3T3 cells were seeded into a 96-well plate at a density of 1×10⁴ cells/well. After 24 h of incubation at 37°C and 5% CO₂, cells were treated with fresh medium containing a series concentration of RGD-KLA-Lips. Cells treated with blank culture media served as a control. After an additional 24 h, 10 μL of CCK-8 was added into each well and cells were further incubated for 2 h at 37°C. The absorbance of the samples was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). The cytotoxicity of PTX formulations, including Taxol (free PTX), RGD/PTX-Lips, KLA/PTX-Lips and RGD-KLA/PTX-Lips, was evaluated using HUVECs and 4T1 cells. Cells were seeded into 96-well plates at a density of 1×10⁴ cells/well and incubated for 24 h at 37°C and 5% CO₂. Cells were then treated using different concentrations of the PTX formulations and evaluated using a CCK-8 assay. The half-maximal inhibitory concentration (IC₅₀) of each treatment was also calculated using Graph Pad Prism 5 software (GraphPad Software, Inc., San Diego, USA).