Background Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope. Results The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 g/ml, while increase in ROS generation and LDH released at 200 and 400 g/ml was observed. Conclusions The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems. (2013). In this technique, the particle sizes were reduced after leaving the homogenising gap by cavitations, particle SC-26196 manufacture collisions, and shear forces. Characterisation of Calcium Carbonate Nanocrystals Particle size and morphology were characterised by transmission electron microscopy (TEM, Hitachi H-7100) and field emission scanning electron microscopy (FESEM, JOEL 7600F) with energy-dispersive X-ray spectroscopy (EDX) as described previously (Kamba < 0.05 was considered significant unless indicated otherwise. Results and Discussion Investigation of nanoparticle cytotoxicity is of paramount importance in various biological fields, including drug discovery, toxicology and ecotoxicology. cell viability (cytotoxicity) assays are used to avoid any unnecessary animal testing, waste of longer time for animal observation after treatment and higher costs materials compared to assays, SC-26196 manufacture which is faster and cheaper to conduct. Selection of the best assay format for meeting a particular need and understanding the endpoint of each assay; what is measured and how the measurement correlates with cell viability are the central key for any analysis. Field Emission Scanning Electron Microscope and Transmission Electron Microscope Characterisation Figures (1a and 1b) demonstrate micrographs of transmission electron microscope (TEM) and field emission scanning electron microscope (FESEM) for the synthesized calcium carbonate Cspg4 nanocrystals, respectively. The images illustrate a uniform particle size distribution with rod-shaped morphology; the synthesized nanoparticles showed individual particle average size of 40 to 60 nm. As shown by the TEM image in Figure (1a), all the particles are within 100 nm, also particle surfaces characterized by FESEM in figure (1b) displayed a rod-shaped particles being the main target of our synthesis. Figure 1 (a and b) is a micrograph images for transmission electron microscope (TEM) and Field Emission scanning electron microscope (FESEM). Cellular Toxicity of CaCO3 nanocrystals against NIH 3T3 Mouse Fibroblasts MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) is a water-soluble tetrazolium salt, and the assay is based on the principle that mitochondrial SC-26196 manufacture dehydrogenase of intact cells may converts the soluble yellow MTT tetrazolium salt into an insoluble purple formazan by cleaving the tetrazolium ring; the formazan product formed is impermeable to cell membranes and therefore only accumulates in healthy cells. Based on the analysis, calcium carbonate nanocrystals show no SC-26196 manufacture apparent toxicity to NIH 3T3 cells, as shown by the MTT results in Figure 2. The results revealed that none of the concentrations (0 to 400 g/ml) used during the study were toxic to the NIH 3T3 fibroblasts cells, and even at the higher concentrations of 200 and 400 g/mL, the percentages of viable cells were 92% and 85%, respectively. The remaining concentrations of calcium carbonate nanocrystals showed no significant difference from the control group (100% viability). Therefore, the result indicates that calcium carbonate nanocrystals did not interfere with the mitochondrial metabolism of the NIH 3T3 fibroblast cells. Figure 2 NIH 3T3 cell viability (MTT) in response to different concentrations of calcium carbonate nanocrystals. Modified Double-Staining Neutral Red/Trypan Blue Exclusion Assay The effect of the calcium carbonate nanocrystals on cell proliferation was analysed by a modified double-staining neutral red/trypan blue method. The results from cells counting demonstrated that the treated cells (10104 cells/mL) showed no significant differences (p>0.05) compared to untreated cells (10.1 104), which indicates that most of the cells were viable; however, reduced number of cells was observed in the treated cells with 200 to 400 g/mL compared to control (untreated) as shown in Figure 3. Therefore, the modified neutral red/trypan blue double-staining method is.