S2). These findings clearly indicate Dabrafenib concentration the
controlled release of iron ion by the chitosan oligosaccharide coating of CSO-INPs, therefore, inducing lesser cellular toxicity in the case of CSO-INPs treated cells. Apoptosis is responsible for multiple alterations in mitochondrial membrane. During apoptosis, mitochondrial phosphatidylserine is externalized from inner surface to the outer surface. Apoptosis is measured in terms of binding of externalized phosphatidylserine to phospholipid binding protein Annexin V conjugated with fluorochromes . Fig. 9 shows that the CSO-INPs treatment causes moderated disintegration in mitochondrial membranes of HeLa, A549 and Hek293 cells as compared to the bare INPs. This data highlights the fact that chitosan coating of iron oxide nanoparticles reduces its apoptotic triggering effects through lesser disintegration of mitochondrial membrane integrity. The loss of mitochondrial membrane potential, a distinctive feature of apoptotic cell, is analysed by cationic carbocyanine dye JC-1. In a normal cell, JC-1 dye is present in monomeric form in cytosol and emits green fluorescence, and accumulate as aggregates in mitochondria emitting red fluorescence. Whereas in mitochondrial
membrane disintegrated apoptotic cell, JC-1 retains its monomeric form in mitochondria and emits green fluorescence only  and . Treatment of iron oxide nanoparticles progressively dissociates mitochondrial potential and increases JC-1 green fluorescence without a corresponding increase in JC-1 red fluorescence INK 128 molecular weight in HeLa, A549 and Hek293 cells, whereas moderate JC-1 red fluorescence was observed in CSO-NPs treated cells in Fig. 10. Thus results suggest that the formation of monomer of JC-1 is high in iron oxide nanoparticles treated HeLa, A549 and Hek293 cells, with respect to CSO-NPs indicating that INPs toxicity may be reduced due to coating of chitosan oligosaccharide. DCFH-DA assay for ROS generation analysis revealed that
Dichlorofluorescein (DCF) production is high in iron oxide nanoparticles treated Hek293, A549 and HeLa cells with respect to CSO-INPs treated cells in Fig. 11. Production of highly fluorescent DCF in INPs treated cells may be attributed to the oxidation Tenoxicam of non-polar dye DCFH-DA by apoptosis induced intracellular ROS and other peroxides. In a non-apoptotic cell DCFH-DA converts to its non-fluorescent, non-polar derivative DCFH by the action of cellular esterase . Dihydroethidine (HE) probe is oxidized into red fluorescent product ethidium in the presence of superoxide anion. This action has been associated with mitochondrial uncoupling and increased ROS production . Interaction of ethidium to DNA is inferred with higher red fluorescence in INPs treated cell compared to CSO-INPs treated HeLa, A549 and Hek293 cells in Fig. S3 (Supplementary data).