Nano-Graphene Oxide for Cellular Imaging and Drug Delivery

Abstract

Two-dimensional graphene offers interesting electronic, thermal, and mechanical properties that are currently being explored for advanced electronics, membranes, and composites. Here we synthesize and explore the biological applications of nano-graphene oxide (NGO), i.e., single-layer graphene oxide sheets down to a few nanometers in lateral width. We develop functionalization chemistry in order to impart solubility and compatibility of NGO in biological environments. We obtain size separated pegylated NGO sheets that are soluble in buffers and serum without agglomeration. The NGO sheets are found to be photoluminescent in the visible and infrared regions. The intrinsic photoluminescence (PL) of NGO is used for live cell imaging in the near-infrared (NIR) with little background. We found that simple physisorption via pi-stacking can be used for loading doxorubicin, a widely used cancer drug onto NGO functionalized with antibody for selective killing of cancer cells in vitro. Owing to its small size, intrinsic optical properties, large specific surface area, low cost, and useful non-covalent interactions with aromatic drug molecules, NGO is a promising new material for biological and medical applications.

Figure 1

Synthesis and pegylation of nano-graphene oxide. (a) Schematic illustration of pegylation of graphene oxide by PEG–stars. (b), (c) AFM images of GO and NGO–PEG, respectively. (d) IR spectra of GO, GO–COOH, and NGO–PEG (the black arrow indicates the characteristic amide-carbonyl vibration)

Figure 2

Optical properties of nano-graphene oxide sheets. (a) UV-vis-NIR absorbance spectra of GO, GO–COOH, and NGO–PEG solutions with 0.01 mg/mL graphitic carbon (1 cm optical path). Inset: a photograph of GO and NGO–PEG solutions at the same graphitic carbon concentration to show the visible color difference. (b) Fluorescence of GO (black curve) and NGO–PEG (red curve) in the visible range under an excitation of 400 nm. (c), (d) Photoluminescence excitation (PLE) spectra of GO & NGO–PEG with 0.31 mg/mL graphitic carbon in the IR region (1 mm optical path). The emission intensity at various emission wavelengths (x-axis) is represented by the color scheme shown as a function of excitation wavelength (y-axis). The data were obtained after normalization to detector sensitivity and absorbance curves. (e) A photograph of an ultracentrifuge tube after density gradient separation of NGO–PEG. (f)–(h) AFM images of NGO–PEG fractions (F5, F13, and F23) at different locations in the centrifuge tube as labeled in (e)

Figure 3

Nano-graphene for targeted NIR imaging of live cells. (a) A schematic drawing illustrating the selective binding and cellular imaging of NGO–PEG conjugated with anti-CD20 antibody, Rituxan. (b) NIR fluorescence image of CD20 positive Raji B-cells treated with the NGO–PEG–Rituxan conjugate. The scale bar shows the intensity of total NIR emission (in the range 1100–2200 nm). Images are false-colored green. (c) NIR fluorescence image of CD20 negative CEM T-Cells treated with NGO–PEG-Rituxan conjugate. (d) Mean NIR fluorescence intensities in the image area for the both the positive (Raji) and negative (CEM) cells treated by NGO–PEG–Rituxan conjugate

Figure 4

Nano–graphene oxide for targeted drug delivery: (a) A schematic illustration of doxorubicin (DOX) loading onto NGO–PEG–Rituxan via π-stacking; (b) UV-vis-NIR absorbance spectra of NGO–PEG and NGO–PEG/DOX. DOX loading on NGO–PEG was evidenced by a strong absorption peak centered at ∼490 nm. The reddish color of the DOX loaded NGO–PEG is seen in the solution (see inset); (c) Retention of over time of DOX on NGO-PEG in buffers at pH 5.5 and 7.4; (d) In vitro toxicity test at 2 µmol/L and 10 µmol/L DOX concentration to show Rituxan selectively enhanced doxorubicin delivery into Raji B-cells by comparing NGO–PEG–Rituxan/DOX with free DOX, a mixture of DOX with NGO–PEG, and a mixture of DOX, Rituxan and NGO–PEG. The viable cell percentage was measured by the MTS assay