For further evaluation of ROS production, HeLa, A549 and Hek293
cells (1 × 105 cells/well) were seeded into 24-well plates and allowed to adhere in 24 h. After 24 h, fresh media was supplemented with 4 μg/μl iron oxide nanoparticles and chitosan oligosaccharide coated iron oxide nanoparticles (CSO-INPs) respectively. selleck compound Cells were trypsinized with 1× trypsin–EDTA, and centrifuged at 1000 rpm for 5 min. Cells were washed twice with 1× PBS buffer. Cells were re-suspended in HBSS (Hanks’ balanced salt solution) buffer containing the fluorescence probes DHE (2.5 μM). Cells were incubated at 37 °C for 20–30 min in dark and washed with 1× PBS buffer [29]. Finally, fluorescence spectrum was measured by flow cytometry (BD Biosciences) at 488 nm excitation and emission at 620 nm wavelength with 10,000 events of each sample. Fluorescence spectra were analyzed by FCS 4 Express Flow Cytometry software.
Significance of the toxicity of iron oxide nanoparticles (INPs) and chitosan oligosaccharide coated iron oxide nanoparticles (CSO-INPs) in MTT assay was analyzed by Student’s t-test. Each experiment, with six in replicates, was performed in at least three independent cell culture preparations. The t-test was used to evaluate the difference in means between groups with a conventional threshold p-value for statistical significance defined as *p < 0.05. Synthesized Fe3O4 nanoparticles were found to be monodisperse and spherical in shape having a mean diameter of 6 ± 1.2 nm in Fig. 1(a). The TEM see more image of Fe3O4-chitosan nanoparticles (CSO-INPs) has been shown in Fig. 1(b). The structures of chitosan oligosaccharide
coated iron oxide nanoparticles were observed bigger in size with a mean diameter of 8 ± 2.7 nm. TEM image clearly indicates that the surface modification process did not cause significant change in the size of the particles. However, a little aggregation was observed in the Fe3O4-CSO nanoparticles, this may be due to higher molecular weight of chitosan oligosaccharide used for the synthesis [22], [32] and [33]. Fig. 2(a) shows X-ray diffraction (XRD) pattern of synthesized iron nanoparticles exhibiting peaks at 2θ at 30.1, 35.5, 42.6, 53.6, O-methylated flavonoid 57.0 and 62.8 which can be assigned to diffraction of the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) planes, respectively of spinal structured magnetite nanoparticles (JCPDS card no. 82-1533). It is to be noted that the coating process did not result in the phase change of Fe3O4. The broad reflection planes can be attributed to the nanosize of the iron oxide nanoparticles [34]. The XRD pattern CSO-INPs exhibited its two characteristic peaks at 2θ = 20.1, 30.1, 35.5 and 62.8 in Fig. 2(b). Presence of characteristic peak at 2θ = 20.1 for chitosan oligosaccharide along with 2θ = 30.1, 35.5 and 62.8 associated with the iron oxide nanoparticles confirms the coating of chitosan oligosaccharide on iron oxide nanoparticles [8] and [35].