Molecular targeting of bioconjugated graphene oxide nanocarriers revealed at a cellular level using label-free Raman imaging.

Two-dimensional materials as graphene oxide (GO) are able to accommodate labels as well as toxins for diagnostics and therapy, respectively. The transmembrane protein carbonic anhydrase (CA IX) is one of the molecules selectively expressed by tumor cells. Here, we demonstrate bioconjugation of GO to biotinylated M75 antibody highly selective towards CA IX. Based on a model system, binding between the bioconjugated GO-M75 and Madin-Darby Canine Kidney (MDCK) cells was evaluated. As proven by fluorescence-activated cell sorting, higher intake was observed for GO-M75 towards MDCK cells ectopically expressing CA IX protein on their surface when compared to control MDCK. In particular, we were able to localize GO nanocarrier crossing the membrane during endocytosis, thanks to the optical cross-sectioning of living cells in real-time employed the label-free confocal Raman microscopy. The increased affinity of the prepared GO-M75 molecular complexes validates the use of two-dimensional materials for future strategies of targeted cancer treatment.

Label-free tracking of nanosized graphene oxide cellular uptake by confocal Raman microscopy.

Graphene oxide (GO), a partially oxidized two-dimensional allotrope of carbon, is an attractive nanocarrier for cancer diagnostics and therapy. The nanometer-sized GO is known to permeate cell membranes. Herein we studied the cellular uptake pathways of GO nanoflakes by cancer and non-cancerous cell lines. By employing confocal Raman imaging, we were able to track the GO cellular uptake in living cells (C33 and MDCK) without any additional fluorescent or plasmonic labels. This specific progress in label-free Raman imaging of GO facilitates the monitoring of nanoflakes at the cellular level.

Fast low-temperature plasma reduction of monolayer graphene oxide at atmospheric pressure.

We report on an ultrafast plasma-based graphene oxide reduction method superior to conventional vacuum thermal annealing and/or chemical reduction. The method is based on the effect of non-equilibrium atmospheric-pressure plasma generated by the diffuse coplanar surface barrier discharge in proximity of the graphene oxide layer. As the reduction time is in the order of seconds, the presented method is applicable to the large-scale production of reduced graphene oxide layers. The short reduction times are achieved by the high-volume power density of plasma, which is of the order of 100 W cm-3. Monolayers of graphene oxide on silicon substrate were prepared by a modified Langmuir-Schaefer method and the efficient and rapid reduction by methane and/or hydrogen plasma was demonstrated. The best results were obtained for the graphene oxide reduction in hydrogen plasma, as verified by x-ray photoelectron spectroscopy and Raman spectroscopy.

Electrochemical performance of composite electrodes based on rGO, Mn/Cu metal-organic frameworks, and PANI.

Benzendicarboxylic acid (BDC)-based metal-organic frameworks (MOFs) have been widely utilized in various applications, including supercapacitor electrode materials. Manganese and copper have solid diamond frames formed with BDC linkers among transition metals chosen for MOF formation. They have shown the possibility to enlarge capacitance at different combinations of MOFs and polyaniline (PANI). Herein, reduced graphene oxide (rGO) was used as the matrix to fabricate electrochemical double-layer SCs. PANI and Mn/Cu-MOF's effect on the properties of electrode materials was investigated through electrochemical analysis. As a result, the highest specific capacitance of about 276 F/g at a current density of 0.5 A/g was obtained for rGO/Cu-MOF@PANI composite.

A Multifunctional Graphene Oxide Platform for Targeting Cancer.

Diagnosis of oncological diseases remains at the forefront of current medical research. Carbonic Anhydrase IX (CA IX) is a cell surface hypoxia-inducible enzyme functionally involved in adaptation to acidosis that is expressed in aggressive tumors; hence, it can be used as a tumor biomarker. Herein, we propose a nanoscale graphene oxide (GO) platform functionalized with magnetic nanoparticles and a monoclonal antibody specific to the CA IX marker. The GO platforms were prepared by a modified Hummers and Offeman method from exfoliated graphite after several centrifugation and ultrasonication cycles. The magnetic nanoparticles were prepared by a chemical precipitation method and subsequently modified. Basic characterization of GO, such as the degree of oxidation, nanoparticle size and exfoliation, were determined by physical and chemical analysis, including X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM). In addition, the size and properties of the poly-L-lysine-modified magnetic nanoparticles were characterized. The antibody specific to CA IX was linked via an amidic bond to the poly-L-lysine modified magnetic nanoparticles, which were conjugated to GO platform again via an amidic bond. The prepared GO-based platform with magnetic nanoparticles combined with a biosensing antibody element was used for a hypoxic cancer cell targeting study based on immunofluorescence.

d,l-lysine functionalized Fe3O4 nanoparticles for detection of cancer cells.

Amino-modified magnetic nanoparticles were prepared by direct chemisorption of biocompatible d,l-lysine (DLL) on electrostatically stabilized magnetic nanoparticles with the aim to bind specific antibodies (Ab) able to detect cancer cells. The magnetic nanoparticles prepared by coprecipitation were stabilized in an acidic medium. A full optimization study of amino modification performed by UV/Vis spectroscopy and Dynamic Light Scattering measurement (DLS) confirmed an optimal DLL/Fe3O4 weight ratio of 2. The sample was subjected to complex characterizations using different techniques such as UV/Vis, FTIR and X-ray photoelectron spectroscopies (XPS) together with transmission electron microscopy and size/zeta potential measurements. While FTIR spectroscopy, UV/Vis spectroscopy and XPS confirmed the successful amino modification of Fe3O4 nanoparticles, a characterization using a vibrating sample magnetometer (VSM) indicated superparamagnetic behavior in all the prepared samples, suggesting that the coating process did not significantly affect the size and structure of the Fe3O4 nanoparticles. Magnetic nanoparticles with the optimal DLL content were conjugated with the M75 monoclonal antibody specific to carbonic anhydrase IX (CA IX), which is considered one of the best markers of tumor hypoxia and a prognostic indicator of cancer progression. The results demonstrate that all tested cell lines survived and even proliferated in the presence of amino-modified magnetic nanoparticles. Even the tubulin cytoskeletal structure was not disrupted after the exposure of cells to surface-modified magnetic nanoparticles. In contrast, internalization of the antibody-conjugated magnetic nanoparticles led to abrogation of the formation of long and extended microtubules. Finally, the finding supports the view that the M75 antibody conjugated to nanoparticles mediates their specific uptake and intracellular accumulation and that the antibody conjugated magnetic nanoparticles can be potentially used for the selective growth inhibition of CA IX-expressing cells.