br blank wells br Internalization of the nanocarriers br
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 .
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.
3.1. 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 .
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 . 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.