2059148-82-0 br Statistical analysis br All values are expre
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 2059148-82-0 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.
The stability of Dox-NPs-Cet was determined by evaluating the re-leased amount of Cet in its storage buﬀer, 0.01 M Tris-HCl (pH7.4), over a period of 24 h. Samples were respectively incubated at 4 °C or at 37 °C for 0.5 h, 1 h, 2 h, 4 h, 6 h and 24 h. Subsequently the particles were separated by the magnetic separator. Supernatants were collected and measured. As shown in Fig. 3B, whether at 4 °C or at 37 °C, the release of Cet always started from the 4th hour. Between 4 and 24 h, only about 20% of Cet was gradually released from the particles. This suggests that Dox-NPs-Cet are relatively stable in the storage buﬀer.
3.3. Internalization of the nanocarriers
We use TEM to observe whether the particles entered the cells and to study the sub-cellular distribution of these particles. As shown in
Fig. 2. The characterization of Dox-NPs-Cet.
A) Representative transmission electron mi-croscope (TEM) images of (a) dextran-coated nanoparticles (b) Dox-NPs-Cet. B) The hydrodynamic size of Dox-NPs-Cet and that of dextran-coated nanoparticles mea-sured by dynamic light scattering. C) The FTIR spectra of (a) dextran-coated nano-particles (b) Dox-NPs (c) Dox-NPs-Cet. D) The magnetization curves of dextran-coated nanoparticles and Dox-NPs-Cet. E) The Dox-NPs-Cet suspension without (left) and with (right) a magnet in its vicinity.