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  • br Materials and methods br Cetuximab

    2020-08-30


    2. Materials and methods
    Cetuximab (Erbitux®, C225) was purchased from Merck Serono Co., Ltd., Beijing, China. Doxorubicin hydrochloride was supplied by Beijing HVsf United Chemical materials Co., Ltd., Beijing, China. The Cell Counting Kit-8 (CCK-8) a cellular proliferation and cytotoxicity assay was obtained from Dojindo, Japan. The Bradford Protein Assay Kit was obtained from Beijing Solarbio Science & Technology Co., Ltd., China. Cell culture medium (Modified RPMI-1640 Medium) was obtained from GE Healthcare Life Science Hyclone Laboratories, Logan, Utah, USA. Fetal Bovine Serum (FBS) was purchased from Beijing Solarbio Science
    & Technology Co., Ltd., China. Trypsin was obtained from Gibco Co., Ltd., USA. Additionally, Fe3O4 magnetic nanoparticles were synthesized by Xi'an GoldMag Nanobiotech Co., Ltd., China. Dextran-40 was  Journal of Magnetism and Magnetic Materials 481 (2019) 122–128
    purchased from Shanghai Huamao Pharmaceutical Co., Ltd. Iron salts were from Sigma-Aldrich (China) Co., Ltd. NaOH was purchased from Xi’an Fuli Chemical Reagents Co., Ltd., China. T-25 culture flasks, 96-and 6-well plates were obtained from Corning, USA. Protein Molecular Weight markers were purchased from Thermo Fisher Scientific, USA. Coomassie brilliant blue G250 was purchased from Beijing Solarbio Science & Technology Co., Ltd., China. The Magnetic Separator was obtained from Xi'an GoldMag Nanobiotech Co., Ltd., China. Water used in the experiments was de-ionized, and all organic solvents were of analytical reagent grade.
    2.2. Preparation of dextran coated iron oxide nanoparticles.
    Iron oxide magnetic nanoparticles were synthesized by Xi'an GoldMag Nanobiotech Co., Ltd., Xi‘an, China. The preparation of dex-tran-coated nanoparticles was performed according to the method de-scribed earlier but with slight modifications [31]. Briefly, 5 g of dex-tran-40 were dissolved in 50 mL of a 0.5 M NaOH solution. After stirring for 10 min, the dextran-40 was completely dissolved. Subsequently, 1 g iron oxide magnetic particles, 50 mL NaOH and 2.686 g Na2HPO4·12H2O were successively added to this LDN 193189 solution . A black suspension was obtained. In order to remove an excess dextran and inorganic salts, dialysis was performed using membranes with a cut-off value of 10 KDa. This process was performed till the particles were brought to neutral pH. The dextran coated particles were used at room temperature for further characterization and for the experiments.
    2.3. Loading of Doxorubicin on dextran-coated iron-oxide nanoparticles.
    Dox was loaded on dextran-coated iron-oxide nanoparticles as fol-lows: 15 mg of Dox dissolved in 15 mL of water was added into a flask containing 150 mg of the dextran-coated iron oxide particles under stirring (180 rpm). The reaction was continued at 37 °C for at least 24 h. Dox loaded nanoparticles (Dox-NPs) were separated by a magnetic se-parator. This instrument was applied for a period of three minutes and the loaded particles were stored at 4 °C for further use and character-ization. The amount of Dox loaded was equal to the difference between the amount of free Dox before and after LDN 193189 conjugation. The concentration of free Dox was monitored with UV–Vis spectrometry at 480 nm using a Shimadzu Corporation, UV-2550 Spectrophotometer.
    2.4. Conjugation of Cetuximab on Dox-NPs
    A quantity of 0.05 mL of a solution containing 100 μg Cet was slowly added into 1 mL of a solution containing 1 mg of Dox-NPs under continuous stirring for a period of 2 h at ambient temperature. The Cet and Dox loaded dextran coated iron oxide nanoparticles (Dox-NPs-Cet) were separated using a magnetic separator for a period of three minutes and stored at 4 °C for further use and characterisation. The amount of Cet loaded was equal to the difference between the amount of free Cet before and after conjugation. The concentration of free Cet was mon-itored by the Brandford method. The absorbance was read on an ELx-800 Universal Microplate Reader (BIOTEK Instrument, Vermont, USA) at 595 nm. r> 2.5. Investigation of the stability of the Dox-NPs-Cet.
    In order to investigate the stability of the Cet conjugated on the Dox loaded dextran-coated iron-oxide nanoparticles in 0.01 M Tris-HCl (pH 7.4) at two different temperatures, 1 mg of the samples were cleaned using a magnetic separator and resuspended in 1 mL 0.01 M Tris-HCl (pH 7.4). The samples were then, kept at 4 °C in the refrigerator or at 37 °C in an incubator. Supernatants were taken after 0, 0.5, 1, 2, 4, 6 and 24 h. The magnetic separator was used to assure the absence of magnetic particles. Absorbance in the purified supernatant was mea-sured at 595 nm (free Cet).