High levels of reactive oxygen species in gold nanoparticle-targeted cancer cells following femtosecond pulse irradiation.

Cell cultures

BJAB and K562 cells were grown at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma) and DMEM medium (Invitrogen), respectively, supplemented by 2 mM glutamine, 5 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum. Cells were maintained at a concentration below 10 6 cells per ml to allow logarithmic growth. MDA-MB-468 cells were grown at 37°C and 5% CO 2 in DMEM medium (Invitrogen) supplemented by 5 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum. For contradicting the effect of ROS, cell cultures were supplemented with fresh medium containing 500 μM ascorbic acid two hours prior to irradiation.

Nanoparticle preparation

Gold nanoparticles were prepared using a citrate reduction protocol 52 resulting in an average particle diameter of 20 nm. Anti-CD20 (Rituximab, Roche Israel) coating of the gold nanoparticles was carried out according to Weiss et al. 53 . Anti-EGFR (Lab Vision, clone designation EGFR.1) coating was achieved by incubating anti-EGFR with OPSS-PEG2000-NHS (Jenkem) for 1 h in a mole ratio of 1:2500, followed by additional 1 h incubation with gold nanospheres in a mole ratio of 1:10 6 nanospheres: OPSS-PEG2000-NHS.

Cell targeting by nanoparticles

BJAB cells (10 6 cells per ml) were incubated for 15 min at 37°C with 4 × 10 10 anti CD20-coated gold nanoparticles per ml. Cells were washed off unbound gold nanoparticles (three PBS washes) prior to laser irradiation. A co-culture of 1:1 ratio BJAB:K562 cells was incubated under the same conditions as described above. MDA-MB-468 cells were incubated for 60 min at 37°C with 2 × 10 11 anti-EGFR-coated gold nanoparticles per ml. Incubation times for each cell line were set for reaching saturation of the number of nanoparticles attached to each cell.

Fluorescence labeling

In the co-culture experiment, the nuclei of the BJAB cells were stained blue using 1.3 μg/ml Hoechst (Aldrich) for 4 minutes at 37°C, followed by 3 PBS washes. Cells were stained for cellular ROS by incubation with 20 μM H 2 DCFDA (Invitrogen) for 5 min at 37°C, or by incubation with 10 μM CellROX™ deep red for 1 h at 37°C. Fluorescence microscopy of the ROS levels was conducted approximately 90 min post-irradiation. The detection of intracellular ROS is highly sensitive to the physiology and metabolism of the cells; hence, different ROS markers that operate through different mechanisms are required for avoiding false positive results. While high levels of ROS in BJAB cells were effectively detected using both H 2 DCFDA and CellROX markers, ROS in MDA-MB-468 cells could not be detected by the H 2 DCFDA marker, probably due to absence of cellular esterases required for activating fluorescence 32 33 . Staining for necrosis and apoptosis was carried out using 1 μg/ml Propidium iodide (Sigma) and Annexin V kit (Roche), respectively. Mitochondrial oxidative activity was detected by incubating the cells with 200 nM MitoTracker red (CM-H 2 XRos; Invitrogen) for 25 min at 37°C.

Actinomycin D treatment of MDA-MB-468 cells

Cells were incubated with 10 μg/ml Actinomycin D for 2 h, followed by three PBS washes.

Laser pulse irradiation

A beam from a Ti:sapphire oscillator (Tsunami, Newport Corp.) was amplified (Spitfire Pro XP) and wavelength-tuned to 550 nm using an optical parametric amplifier (OPA; Topas-C). Pulse duration was 50 fs, at 1 kHz repetition rate. Cells were irradiated within eight-well chamber slides (Lab-Tek II, Thermo-scientific) placed within a microscope stage incubator (Okolab Inc.) at controlled temperature and CO 2 concentration. The irradiated area of each cell culture was approximately 100 mm 2 and 16 mm 2 for the BJAB and MDA-MB-468 cells, respectively, using repeated pulse irradiation at an array of approximately 300 μm diameter spots. Single pulse fluence was either 16.5 mJ/cm 2 (3.3 × 10 11 W/cm 2 ) or 24.5 mJ/cm 2 (4.9 × 10 11 W/cm 2 ) for the BJAB and MDA-MB-468 cells, respectively. Multiple pulse irradiations per spot was achieved by tuning the beam scanning rates so that each culture location was irradiated by the desired number of overlapping spots.

Data analysis

Selected images of BJAB and MDA-MB-468 cells, acquired 90 min after irradiation, were used to estimate the percentage of cells with high levels of ROS using NIS-Elements Advanced Research (Nikon) software. The percentage of cells with ROS levels above twice the basal level of the control cells (nanoparticle-free, non-irradiated) was evaluated for different irradiation parameters using manual cell counting. In cultures where cell fusion was abundant, each nucleus within the fused cells was considered as a single ROS-expressing cell, resulting in up to 10% bias of our estimation toward higher abundance of cells affected by ROS. Selected images of MDA-MB-468 cells, acquired 22 h and 50 h post irradiation were used to estimate the percentage of dead (necrotic, apoptotic or cell cadavers) cells for different irradiation parameters using manual counting. The statistical significance of the results was assessed using a two-proportion z-test.

Supplementary Material Supplementary Information Dataset 1-7