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  • br blank wells br Internalization of the nanocarriers br


    blank wells.
    2.10. Internalization of the nanocarriers
    To further confirm the intracellular uptake and localization of the nanocarriers, A549 Chloramphenicol 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 [35].
    2.11. Statistical analysis
    All values are expressed as the mean ± standard deviation (SD). GraphPad Prism 5.0 was used for the analysis of the experimental data. Statistical analysis of the data was performed using the T-test or the One-Way ANOVA-Ordinary test. A P value of less than 0.05 was con-sidered statistically significant in all cases.
    3. Results
    3.1. Physicochemical properties of compound particles
    The properties of dextran-coated nanoparticles and Dox-NPs-Cet were measured by TEM and Dynamic light scattering. TEM pictures show that Dox-NPs-Cet have better-dispersed than dextran-coated na-noparticles (Fig. 2A-a, b). On the basis of dynamic light scattering (DLS) measurements, the hydrodynamic diameter of Dox-NPs-Cet was de-termined as 144.5 nm with perfect size distribution, which is larger than that of the unmodified dextran-coated nanoparticles of 116.1 nm. This is because coating Cet on the surface of dextran-coated nano-particles leads to a layer of adsorbed Cet protein which contributes to the hydrodynamic diameter. The diameter of Dox-NPs-Cet was in the appropriate range for a drug carrier [34].
    To investigate whether the particles have been successfully coated, Dox-NPs-Cet were analyzed by FTIR. The FTIR spectra of dextran-coated nanoparticles, Dox-NPs and Dox-NPs-Cet are depicted in Fig. 2C. The FTIR spectrum for dextran-coated Fe3O4 particles shows that the characteristic absorption bands at 583 cm−1 belong to the Fe-O bonds, the absorption peaks at 3600–3200 cm−1 and 1150–1085 cm−1 are attributed to the O–H and C–O–C bonds (Fig. 2C-a). In addition, Fig. 2C-
    c shows the typical absorption peaks of Dox-NPs-Cet in the range of 1600–1700 cm−1 where the amide group absorbs [1]. The results in-dicate that dextran-coated Fe3O4 nanoparticles were successfully loaded with Dox and Cet. Furthermore, the amounts of Dox and Cet loaded on 1 mg dextran-coated Fe3O4 nanoparticles were measured. They were 41.5 ± 0.84 µg (Dox) and 36.24 ± 0.184 µg (Cet).
    In further analyses, the magnetic properties of the nanoparti-cles were studied with VSM (Vibrating Sample Magnetometry) at room temperature. As shown in Fig. 2B, the magnetization of dextran loaded particles was 10.0 emu/g. After conjugation with Dox and Cet, it was 4.6 emu/g. This probably relates to the increase of the coating layer and the decrease of the mass percentage of iron oxide. Although the mag-netization was reduced, the nanoparticles could still be successfully controlled using magnetic forces and they had a better magnetic re-sponsiveness (Fig. 2E). When removed with a magnetic field, Dox-NPs-Cet nanoparticles remained well dispersed without a magnetic memory. This is very beneficial for the separation operation during co-conjugate preparation.
    3.2. The result of SDS-PAGE and the stability of Dox-NPs-Cet
    SDS-PAGE analysis was used to verify whether Cet was indeed loaded on the surface of the Dox-NPs. Fig. 3A shows the SDS gel con-firming the presence of the Cet antibody on the Dox-NPs-Cet. Two bands are present in lane 2 where Cetuximab alone has been applied. In lane 4, where Dox-NPs-Cet have been applied, both the heavy chain and the light chain of the antibody can also be distinguished. The bands in lane 2 and lane 4 have the same molecular weights. The results of SDS-PAGE indicate a successful coating of Dox-NPs with Cet.