Selim et al. Nanoscale Research Letters 2011, 6:528 http://www.nanoscalereslett.com/content/6/1/528 Page 6 of 9 Figure 4 Particle size distributions of (a) mercaptoacetic acid- coated CSNPs and (b) ICSNPs. As measured by dynamic light scattering. serum. Cell proliferation in the presence of CSNPs, PCSNPs, and ICSNPs and in the absence of nanoparti- cles was further visualized using an optical microscope. As a result, cell proliferation in the presence of ICSNPs Figure 5 Proliferation of human fibroblasts after 6 and 24 h of incubation. In a dish containing CSNPs, PCSNPs, and ICSNPs and in a polystyrene culture dish, as measured by BrdU assay. was found to be higher than that in the presence of CSNPs and PCSNPs, as shown in Figure 6. Cellular cytotoxicity The status of the ‘’Live/Dead’’ dye-stained fibroblasts cultured in the presence of CSNPs, PCSNPs, and ICSNPs and in the absence of nanoparticles for 2 days is shown in Figure 7. Using this qualitative method, live and dead cells were stained green and red, respectively, under a fluorescence microscope. The color of the cells cultured on a PS dish and with ICSNPs was green, indi- cating good viability. On the other hand, when cultured with CSNPs and PCSNPs, the green color was partially mixed with red, showing that some parts of the cells were dead. Possible explanations are: (1) toxic cadmium ion (Cd+2) release from CSNPs due to surface oxidation causes cell death [4,33]; (2) reactive oxygen species (ROS) react with cellular biomolecules, resulting in damage, degradation, and finally loss of function [34,35]; (3) nanoparticles are taken up by the cells as a result of endocytosis, which results in disruption of the cell mem- brane [36]; or (4) weak cell adhesive interactions with CSNPs promote apoptosis (programmed cell death) [36]. Since mercaptoacetic acid is the least solubilizing ligand, and it alone could not protect CSNPs from surface oxi- dation and diffusion of Cd+2 ions from CSNPs over a longer period, it is difficult to make CSNPs biological inert. Therefore, most of the cells died in the presence of CSNPs [4,33,34]. Again, the cell viability of PCSNPs was moderately low due to the surface immobilization conferred by biocompatible PEG, which reduced the release of cadmium ion (Cd+2) and formation of ROS. On the other hand, ICSNPs revealed no cytotoxic effects on cells for up to 2 days. This increased cell viability can be explained by a nutrient effect [36,37]. Besides, the low toxicity of nanoparticles immobilized with insulin may be attributed to the fact that these ligands act as