Induced Nucleation of Biomimetic Nanoapatites on Exfoliated Graphene Biomolecule Flakes by Vapor Diffusion in Microdroplets

The nucleation of apatite nanoparticles on exfoliated graphene nanoflakes has been successfully carried out by the sitting drop vapor diffusion method, with the aim of producing cytocompatible hybrid nanocomposites of both components. The graphene flakes were prepared by the sonication-assisted, liquid-phase exfoliation technique, using the following biomolecules as dispersing surfactants: lysozyme, L-tryptophan, N-acetyl-D-glucosamine, and chitosan. Results from mineralogical, spectroscopic, and microscopic characterization (X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman, Variable pressure scanning electron microscopy (VPSEM), and transmission electron microscopy (TEM)) indicate that flakes were stacked in multilayers (> 5 layers) and most likely intercalated and functionalized with the biomolecules, while the apatite nanoparticles were found forming a coating on the graphene surfaces. It is worthwhile to mention that when using chitosan-exfoliated graphene, the composites were more homogeneous than when using the other biomolecule graphene flakes, suggesting that this polysaccharide, extremely rich in –OH groups, must be arranged on the graphene surface with the –OH groups pointing toward the solution, forming a more regular pattern for apatite nucleation. The findings by XRD and morphological analysis point to the role of “functionalized graphene” as a template, which induces heterogeneous nucleation and favors the growth of apatite on the flakes’ surfaces. The cytocompatibility tests of the resulting composites, evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose–dependent manner on GTL-16 cells, a human gastric carcinoma cell line, and on m17.ASC cells, a murine mesenchymal stem cell line with osteogenic potential, reveal that in all cases, full cytocompatibility was found.