Rapid detection of oral cancer using Ag-TiO2 nanostructured surface-enhanced Raman spectroscopic substrates.

The unique vibrational signatures of the biochemical changes in tissue samples may enable the Raman spectroscopic detection of diseases, like cancer. However, the Raman scattering cross-section of tissues is relatively low and hence the clinical translation of such methods faces serious challenges. In this study, we report a simple and efficient surface-enhanced Raman scattering (SERS) substrate, for the rapid and label-free detection of oral cancer. Raman active silver (Ag) surfaces were created on three distinct titania (TiO2) hierarchical nanostructures (needular, bipyramidal and leaf-like) by a process involving a hydrothermal reaction, followed by the sputter deposition of Ag nanoparticles (average size: 30 nm). The resulting SERS substrate efficiencies, measured using crystal violet (CV) as an analyte molecule, showed a highest analytical enhancement factor of ∼106, a detection limit ∼1 nM and a relative standard deviation of the Raman peak maximum of ∼13% for the nano-leafy structure. This substrate was used to analyze tissue sections of 8 oral cancer patients (squamous cell carcinoma of tongue) comprising a total of 24 normal and 32 tumor tissue sections and the recorded spectra were analyzed by principal component analysis and discriminant analysis. The tissue spectra were correctly classified into tumor and normal groups, with a diagnostic sensitivity of 100%, a specificity of 95.83% and the average processing time per patient of 15-20 min. This indicates the potential translation of the SERS method for the rapid and accurate detection of cancer.

Confocal Raman imaging study showing macrophage mediated biodegradation of graphene in vivo.

This study is focused on the crucial issue of biodegradability of graphene under in vivo conditions. Characteristic Raman signatures of graphene are used to three dimensionally (3D) image its localization in lung, liver, kidney and spleen of mouse and identified gradual development of structural disorder, happening over a period of 3 months, as indicated by the formation of defect-related D'band, line broadening of D and G bands, increase in ID /IG ratio and overall intensity reduction. Prior to injection, the carboxyl functionalized graphene of lateral size ∼200 nm is well dispersed in aqueous medium, but 24 hours post injection, larger aggregates of size up to 10 µm are detected in various organs. Using Raman cluster imaging method, temporal development of disorder is detected from day 8 onwards, which begins from the edges and grows inwards over a period of 3 months. The biodegradation is found prominent in graphene phagocytosed by tissue-bound macrophages and the gene expression studies of pro-inflammatory cytokines indicated the possibility of phagocytic immune response. In addition, in vitro studies conducted on macrophage cell lines also show development of structural disorder in the engulfed graphene, reiterating the role of macrophages in biodegradation. This is the first report providing clear evidence of in vivo biodegradation of graphene and these results may radically change the perspective on potential biomedical applications of graphene.

Hemocompatibility and macrophage response of pristine and functionalized graphene.

Graphene and its derivatives are being proposed for several important biomedical applications including drug delivery, gene delivery, contrast imaging, and anticancer therapy. Most of these applications demand intravenous injection of graphene and hence evaluation of its hemocompatibility is an essential prerequisite. Herein, both pristine and functionalized graphene are extensively characterized for their interactions with murine macrophage RAW 264.7 cells and human primary blood components. Detailed analyses of the potential uptake by macrophages, effects on its metabolic activity, membrane integrity, induction of reactive oxygen stress, hemolysis, platelet activation, platelet aggregation, coagulation cascade, cytokine induction, immune cell activation, and immune cell suppression are performed using optimized protocols for nanotoxicity evaluation. Electron microscopy, confocal Raman spectral mapping, and confocal fluorescence imaging studies show active interaction of both the graphene systems with macrophage cells, and the reactive oxygen species mediated toxicity effects of hydrophobic pristine samples are significantly reduced by surface functionalization. In the case of hemocompatibility, both types of graphene show excellent compatibility with red blood cells, platelets, and plasma coagulation pathways, and minimal alteration in the cytokine expression by human peripheral blood mononuclear cells. Further, both samples do not cause any premature immune cell activation or suppression up to a relatively high concentration of 75 µg mL(-1) after 72 h of incubation under in vitro conditions. This study clearly suggests that the observed toxicity effects of pristine graphene towards macrophage cells can be easily averted by surface functionalization and both the systems show excellent hemocompatibility.