Nanoparticles (NPs) internalization process in living cells has large perspectives for drug-delivery applications, but the efficient cellular uptake is still a remarkable challenge. Recently, it has been demonstrated that nanographene oxide (nGO) particles are massively internalized into the cell cytoplasm, opening the way of using graphene family materials for the above goal. Internalized nGO can interact with intracellular elements, thus modifying the life cycle pathways. Therefore, there is a great interest in studying the cellular uptake process and the volumetric distribution of nGO inside the cells. He we report on the use of holographic microscopy for quantitatively evaluating the nGO cellular uptake in both 2D and 3D. In particular, quantitative phase images of adherent cells with internalized nGO are used to measure the cells biovolume variation in time, while tomographic reconstructions of cells in flow cytometry condition are exploited to visualize in 3D the distribution of nGO within the cell's cytoplasm. The study is conducted on NIH-3T3 cells to analyze the effects of nGO in vitro and monitoring the cell culture was for several hours to allow a time-lapse of nGO uptake.
Detection and quantification of intracellular structures is fundamental in biomedical sciences. New emerging inspection tools based on holographic microscopy and quantitative phase imaging can give answers to such critical demands. Holographic tomography (HT) systems are the best candidates for this challenge. Recently, HT has been demonstrated working in flow-cytometry (FC) modality. Results show that the novel HTFC tool is capable to furnish 3D visualization and quantifications of the different intracellular particles. In particular, here we report that exogenous nanographene oxide particles as well as endogenous lipid droplets can be detected, measured, and visualized in each flowing cell by label-free HTFC. This method opens the way for accurate and high-throughput measurements at the 3D single-cell level for different applications such as diagnosis of diseases, development of drug delivery applications, and examination of cell functionalities. Experiments and processing methods will be described, and several examples will be discussed.
Nano graphene-based materials offer interesting physicochemical and biological properties for biotechnological applications due to their small size, large surface area and ability to interact with cells/tissues. Among carbon-based nanomaterials, graphene oxide is one of the most used in biological field. There is an increasing interest in shedding light on the interaction mechanisms of nanographene oxide (nGO) with cells. In fact, the effects on human health of GO, and its toxicological profile, are still largely unknown. Here we show that, by minimizing the oxidation degree of GO, its toxicity is significantly reduced in NIH 3T3 cells. Moreover, we show that mild oxidation of graphene nanoplatelets produces nGO particles, which are massively internalized into the cell cytoplasm. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to analyze cell viability. Transmission electron microscopy (TEM) analysis was performed to evaluate nGO internalization mechanism into the cytoplasm under different oxidation degree and concentrations. For the first time, we evaluated quantitatively, the cell volume variation after nGO internalization in live fibroblasts through a label-free digital holography (DH) imaging technique and in quasi-real-time modality, thus avoiding the time-consuming and detrimental procedures usually employed by electron-based microscopy. In conclusion, here we have demonstrated that DH can be a viable tool to visualize and display 3D distributions of nano graphene oxide (nGO) uptake by fibroblast cells. DH opens the route for high-throughput investigation at single cell level for understanding how in different conditions nanoparticles aggregates distribute inside the cells.
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