Functionalization of Carbon nanotubes. SWCNTs (Aldrich, MO) and MWCNTs (Helix, TX) were coated with phospholipid polyethylene glycol amine (LP) (1,2-distearoyl-sn-glycero-3-phosphoethanolmine-N-[amino(polyethyleneglycol)-2000] (ammonium salt) from Avanti Polar Lipids, Inc., AL) to render CNTs dispersible in aqueous medium. To accomplish this SWCNTs (1 mg) or MWCNTs (1 mg) were added to an aqueous solution of LP (1 mg ml-1) and sonication was carried out with the help of an ultra sonicator (Qsonica, CT) for 3 h. This process resulted in aqueous dispersed CNTs. To remove any uncoated SWCNTs or MWCNTs, the LP coated CNTs were centrifuged for 2 h at 10,000 rpm. After the centrifugation the bare uncoated nanotubes settled at the bottom of the vial. The supernatant was recovered, and unreacted LP was removed using a 5K MW centricon filter and stored for later use.
FTIR of Functionalized CNTs. Fourier transform infrared (FTIR) spectroscopy of LP coated SWCNT and MWCNT nanoconjugates, and of pristine SWCNT and MWCNT controls, was done using KBr pellets of each. The wave numbers of the transmittance of each sample were recorded using a Perkin-Elmer spectrum GX spectrophotometer.
Fluorescence Spectrometry. Fluorescence spectrometer was used to assess the presence of FITC on the LP coated SWCNTs or MWCNTs. For this fluorescence spectra of FITC conjugated to LP coated SWCNTs or MWCNTs (with pristine SWCNTs and MWCNTs as controls) were obtained using a Perkin-Elmer LS55 fluorescence spectrometer.
Atomic Force Microscope analysis of CNT nanoconjugates.
Samples for AFM were prepared and analyzed as described with modifications 44-45
. Briefly, biological AFM imaging of the carbon nanotube samples was carried out under a range of deposition conditions in fluid, rinsed with deionized water and dried under a gentle flow of N2
or Ar gas, using a gentle tapping-mode on a PicoForce Multimode AFM (Bruker, CA) consisting of a Nanoscope® V controller, a type E scanner head, and a sharpened TESP-SS (Bruker, CA), OMCL (Bruker, CA), or similar AFM cantilever. For either SWCNT or MWCNT sample visualization, suitable surface attachment of the respective sample was achieved readily by 5 min incubation of 20 μL of the fluid suspension on freshly-peeled mica disks of 12 mm diameters. The excess fluid was blown away by an inert gas gust, or blotted with filter paper, followed by two rinses each with 30 μL of deionized water to remove salt deposits and by a complete drying of the sample surface under gentler gas flow. The sample was then sealed into the instrument compartment (dehumidified by Drierite® particles) and imaged via standard optimizations. AFM images were evaluated within the Nanoscope software (version 7.2, Bruker), and exported to Image J (version 1.4x, NIH, Bethesda, MD) for further analyses and display.
Zeta Potential. To find the surface charge of LP coated SWCNTs and MWCNTs, zeta potential analysis was carried-out using a Zetasizer Nano series (Zen3600) from Malvern with Zetasizer software 6.0 as the interface.
Raman analysis of CNT nanoconjugates. Raman characterization was used for the analysis of the functionalized LP coated SWCNT and MWCNT samples for specific signature peaks along with controls consisting of pristine SWCNTs and MWCNTs. Raman characterization of aqueous dispersed CNTs was done by using characteristic SWCNT/MWCNT Raman signature peaks (G and D bands). Raman spectra were recorded on an iRaman 785 (B&W Tek, Inc, DE) spectrometer equipped with a microscope attachment, and laser spot size was focused to 1 μm diameter (×100 objective), with a power output of 35 mW. The excitation source was an argon ion laser, 785 nm.
2D and 3D cell culture. For 2D cell culture, OVCAR8 ovarian cancer cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37°C in 95% air/5% CO2. Freshly plated cells were grown overnight, to 50-70% confluency prior to incubation for 3 hrs with 200 μL RPMI containing either LP coated SWCNTs or MWCNTs, and after 3 washes with PBS the treated cells were incubated further in fresh RPMI containing serum and carefully monitored microscopically (Axiovert 200m; Carl Zeiss). For 3D cell cultures, OVCAR8 cells were trypsinized, collected, mixed with matrigel, and plated in 8 well chambered cultured slides, which had been previously coated with a thin layer of matrigel. The matrigel mixed cells were observed for the spheroid formation.
Cell proliferation (MTT) assay. Cells were grown to 50-70% confluency overnight in 96 well plates. Next, the media was aspirated and the cells incubated with fresh media containing either LP coated SWCNTs or MWCNTs at various concentrations along with control cells for 48 h. Post treatment the cells were washed 2 times in PBS, and then incubated for an additional 24 h in fresh media. MTT was assessed using a CellTiter 96 AQ One Solution Cell Proliferation Assay kit (Promega, WI), and measured optically at 570 nm.
TUNEL assay. An APO-BrdU TUNEL (Terminal transferase dUTP nick end labeling) assay kit (Invitrogen, CA) was used to detect cells undergoing apoptosis. Briefly, cells were grown onto 8 well chambered cell culture slides until 50-70% confluent, followed by treatment with either SWCNTs or MWCNTs. Control cells were grown in the absence of CNTs. After treatment, the cells were washed in PBS (3x for 2 min each) and incubated in fresh media for 1 h. Next, cells were fixed in 3.5 % PBS-formaldehyde for 15 min at RT, rinsed in PBS (3x) and permeabilized in 0.5 % PBS-Tween 20 for 5 min at RT followed by DNA end-labeling for 1 h at 37 °C. Then, the labeled cells were washed, and incubated further with 95 μL antibody staining solution (AlexaFluor 488-conjugated anti-BrdU) for 30 min at 37 °C, followed by additional washes (3x). Fluorescence image acquisitions were performed with an Axioplan 2 (Carl Zeiss).
Confocal microscopy. OVCAR8 ovarian cancer cells were grown 50-70% confluent on 8-well chambered LabTek II coverglass, treated with either FITC or Qdot conjugated LP coated SWCNTs or MWCNTs, as described above and, incubated for 3 h. Cells were next washed with PBS (3x) and resuspended in fresh media. Live cell imaging was performed using an inverted Zeiss LSM 700 confocal microscope equipped with a CO2 module, heating unit and heating plate, using a 40x/0.75 M27 EC Plan-Neofluar objective. Imaging was carried out at 37°C in 5% CO2 with cells plated in LabTek II coverglass (Nalge Nunc International, NY). Images were acquired and processed with the Zeiss Zen 2009 image software. The fluorescence micrographs shown are representative of at least three independent experiments. Average fluorescence intensity was quantified using Zen 2009 software.
Transmission electron microscope imaging of SWCNTs/MWCNTs. A specimen of LP-coated SWCNTs or MWCNTs for TEM imaging was prepared by depositing a 3 μL droplet from the aqueous solution onto a Quantifoil grid and letting it dry in air. After adsorption for 3 min, the excess solution was blotted with filter paper, washed with a few 3-μl droplets of de-ionized water in order to remove any dirt, and left to dry. Transmission electron micrographs were recorded by means of a Tecnai TF30 TEM (FEI, Hillsboro, OR) equipped with a Gatan Ultrascan 1000 CCD camera (Gatan, Pleasaton, CA).