Hyclone dmem

RAW 264.7 cells (mouse monocyte macrophages) and 4T1 cells (murine mammary carcinoma cells) were purchased from the American Type Culture Collection. Cells were cultured in HyClone^® DMEM (Thermo Fisher Scientific, Inc.) with 10% FBS containing 1% (v/v) penicillin-streptomycin and 1% (v/v) Fungizone^® antimycotic. Cells were maintained in 37°C humidified air containing 5% CO₂.
1×10⁶ macrophages were seeded in 6-well plates and cultured with 2 ml medium for 3 h. A total of 2 µl IL-4 (SRP3211-20UG, Sigma-Aldrich; Merck KGaA)was then added into the medium, and the cells were cultured for a further 12 h at 37°C to obtain high expression levels of CD206 in the macrophages. Macrophages were subsequently incubated at 37°C with 500 µl CD206-Fe₃O₄-PLGA nanoparticles or with Fe₃O₄-PLGA nanoparticle suspension, for 30 min. Then, the macrophages were washed three times with PBS, incubated with rabbit anti-mouse secondary antibodies labeled with TRITC (1:1,000) at room temperature for 1 h and washed with PBS for a further three times. Fluorescence microscopy was used to observe the targeting capacity of the nanoparticles (magnification, ×400).

LS174T human colon cancer, PC9 human lung cancer, and FaDu squamous cell carcinoma cell lines, which all overexpress epidermal growth factor receptor, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were grown in Hyclone™ DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; JR Scientific, Woodland, CA, USA) and 1% antibiotics (Thermo Fisher Scientific). The medium was changed twice or three times per week. The cells were cultured at 37°C in a 5% CO₂ atmosphere.