br Fig Schematic illustration of the Dox NPs Cet
Fig. 1. Schematic illustration of the Dox-NPs-Cet therapy for non-small cell lung cancer (NSCLC).
2.6. Characterization of the nanocarriers
The particle sizes of the dextran-coated iron oxide particles and of the Cetuximab and Doxorubicin loaded dextran coated iron oxide na-noparticles were determined by using a Dynamic light scattering (DLS) instrument (Malvern Zetasizer Nano-ZS, Malvern Instrument, Worcestershire, UK). The morphology of the nanocarriers was studied by using a Transmission Electron Microscope (TEM) (Hitachi H-600, Hitachi Corporation, Tokyo, Japan). In order to characterise the com-position of the nanocarriers, the presence of iron oxide bonds, the nature of the coating and its bonding on the surface Fourier transform infrared spectra (FTIR) were produced. These were recorded, using the KBr pellet technique, between 4000 and 400 cm−1 on a Nicolet 5700 FTIR spectrometer 60 (Thermo Nicolet 5700, Thermo Nicolet Corporation, Wisconsin, USA). Vibrating sample magnetometry (VSM) was used to measure the magnetic properties of the diﬀerent nano-carriers with a magnetometer (LakeShore-655, Lake Shore Inc., USA), at room temperature.
2.7. Identification of the loaded-Cet using SDS-PAGE.
For SDS-PAGE, analysis, 30 μL of a Cet (2 mg/mL), Dox-NPs (1 mg/ mL) and Dox-NPs-Cet (1 mg/mL) solution were mixed in an Eppendorf tube with 2 μL DTT (2 M) and 10 µL 4 × SDS-PAGE loading buﬀer (1 M Tris-HCl pH 6.8, 10% SDS, glycerol, bromophenol blue). Prior to the electrophoresis, the samples were heated in a boiling water bath for 10 min to assure protein denaturation. Then, 10 µL of the samples were loaded on a 12% SDS-PAGE gel. The same volume of the protein ladder mixture was used as a control. Electrophoresis was run at room tem-perature for 30 min at 80 V and subsequently at 120 V till the color indicator arrived at the bottom of the gel. Afterwards, the resulting gel was stained with Coomassie brilliant blue G250 in order to detect the protein bands.
2.8. Cell lines and cell culture
A549, adenocarcinomic human alveolar basal epithelial cells,
originating from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were kindly provided by Shaanxi Lifegen Co., Ltd Xi’an, China and cultured in T25 culture flasks in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 LDN 193189 using a Thermo Scientific Forma Series Ⅱ Water Jacket CO2 incubator. The culture medium was renewed every 2–3 days and the cells were passaged at 1:2 every 2–3 days following trypsinization with 0.05% trypsin-EDTA.
2.9. In vitro cytotoxicity assays
Viability of A549 cells, cultured in the presence of dextran-coated particles, loaded or not loaded with Doxorubicin or/and Cetuximab, was determined using the CCK-8 assay. The test is based on the trans-formation by living cells of the highly water-soluble tetrazolium salt [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] into a water-soluble formazan dye. The quantity of the formazan dye produced is directly proportional to the number of living cells and has been shown to correlate with [3H]-thymidine incorporation. An A549 cell-suspension containing 5 × 103 cells/100 μL was seeded into wells of 96-well plates at 100 μL per well and incubated for 24 h in modified RPMI-1640. The medium was then removed and various concentrations of dextran-coated particles, loaded or not loaded with Doxorubicin or/and Cetuximab were added to the 96-well plates and incubated for 24 h or 48 h at 37 °C in a 5% CO2 incubator. Various concentrations of Dox were obtained by dilution of Dox-NPs or Dox-NPs-Cet that we prepared at the beginning. After in-cubation, supernatants were removed, and all wells were washed twice with PBS buﬀer. Then, according to the manufacturer‘s operating in-structions, 100 µL of 10% CCK-8 solution was added to each well after which the plate was placed in the incubator for 4 h. Subsequently the absorbance in each well was measured at 450 nm with the use of a microplate reader (Epoch Microplate Spectrophotometer, BioTek Instruments, Inc.). The cell viability rate (%) was calculated using the following formula: Cell viability rate (%) = [(As-Ab]]/[(Ac-Ab)] × 100%, where As is the absorbance of the experimental wells, Ac is the absorbance of the control wells and Ab is the absorbance of the
2.10. Internalization of the nanocarriers
To further confirm the intracellular uptake and localization of the nanocarriers, A549 cells were seeded at a density of 1 × 105 per well in 6-well plates (Corning, USA) and incubated for 24 h. Cells were treated with Dox-NPs-Cet in modified RPMI-1640 at 37 ℃ for 3 h. The cells were then trypsinized with 0.2 mL of a 0.25% trypsin solution per well (0.25%) after which they were washed twice with PBS and harvested. Cell fixation steps and placing on 400 mesh copper grids for TEM were performed according to standard protocols .