After each washing, samples were centrifuged at 50,000 g for 30 min at 4uC. To prepare fluorescently labeled nanotubes, SWCNT were sonicated with either PC or PC/PS liposomes containing NBD-PC or NBD-PS (10% of total phospholipids), respectively. For the PS-coated Annexin V treated SWCNT, PS- coated SWCNT were incubated with Annexin V (25 mg/mg SWCNT) in Annexin V binding buffer for 5 min at room temperature and then washed twice with 25 mM HEPES, pH 7.4 to remove non-bound Annexin V. After each washing, PS-coated/ Annexin V treated SWCNT were centrifuged at 50,000 g for 15 min. To prepare PS/cyt c/SWCNT, nanoparticles (0.3 mg/ ml) were incubated in 25 mM HEPES buffer, pH 7.4 with 50 mM cyt c for 30 min at room temperature. To remove non-bound cyt c, SWCNT were washed twice with 25 mM HEPES buffer pH 7.4 and centrifuged at 50,000 g for 30 min at 4uC. After that, cyt c/ SWCNT were sonicated in the presence of 3 mM PC:PS liposomes (at the ratio of 1:1) 3 cycles for 30 s and then washed 4 times with 25 mM HEPES. After each washing, samples were centrifuged at 50,000 g for 30 min at 4uC. Coated SWCNT were finally suspended in 25 mM HEPES pH 7.4 (containing a transition metal chelator, DTPA (100 mM) to prevent oxidative damage to lipids and protein) using the same volume as the original suspension. Endotoxin content in SWCNT suspensions and its amounts present in the medium during incubations were approximately 300–500 times lower than those causing stimulation of macrophages. Determination of phospholipid content Phospholipids from coated SWCNT were extracted using Folch procedure [34] and separated by one dimensional HPTLC. Spots of PS or PC were visualized by exposure to iodine vapors and compared with authentic standards. Phospholipid phosphorus was determined using sub-micro method [35]. Animals Specific-pathogen-free adult female C57BL/6 mice (7–8 wk) were supplied by Jackson Lab (Bar Harbor, ME) and weighed 20.360.2 g at time of use. Animals were individually housed in AAALAC-approved NIOSH animal facilities in microisolator cages for one week prior to use. Autoclaved Beta Chip bedding (Northeastern Products Corp., Warrensburg, NY) was changed weekly. Animals were supplied with water and Harlan Teklad, 7913, NIH-31 Modified Mouse/Rat Diet, Irradiated (Harlan Teklad, Madison, WI) and housed under controlled light, temperature and humidity conditions. Experiments were conduct- ed under a protocol approved by the Animal Care and Use Committee of the NIOSH. Mice were randomized into three experimental groups treated either with non-coated SWCNT, PC- coated SWCNT or PS-coated SWCNT on day 0. Animals were sacrificed on day 1 following exposures. Particulate Instillation Pharyngeal aspiration was used for particulate administration to C57BL/6 mice. Briefly, after anesthization with ketamine and xylazine anesthesia (62.5 and 2.5 mg/kg, respectively), the mouse was placed on a board in a near vertical position. The animal’s tongue was extended with lined forceps and a suspension of particulates (50 ml, non-coated SWCNT, PC-coated SWCNT or PS-coated SWCNT at a dose of 40 mg/mouse) was placed in the posterior of pharynx. The tongue was held until the suspension was aspirated into the lungs. All mice in particle and PBS groups survived this exposure procedure. This technique provides good distribution of particles widely disseminated in a peri-bronchiolar pattern within the alveolar region [36]. Animals treated with the Targeting Phagocytes by PS-CNT particulates recovered easily after anesthesia with no behavioral or negative health outcomes. Bronchoalveolar lavage Mice were weighed and euthanized with intraperitoneal injection of sodium pentobarbital (SPB, Fort Dodge Animal Health, Fort Dodge, Iowa) (.100 mg/kg). The trachea was cannulated with a blunted 22 gauge needle, and BAL was performed using cold sterile Ca2++Mg 2+-free PBS at a volume of 0.9 ml for first lavage (kept separate) and 1.0 ml for subsequent lavages. Approximately 5 ml of BAL fluid per mouse was collected and pooled in sterile centrifuge tubes. Pooled BAL cells were washed in Ca+2+Mg+2-free PBS by alternate centrifugation (8006g for 10 min at 4uC) and resuspension. Cells Primary microglia was isolated from brains of postnatal day 5 rats as described [37]. More than 80% of the cell population was represented by microglia as evidenced by immunostaining with the microglia CD11 (mouse monoclonal anti-CD11 antibody were from Novus Biologicals, Inc. (Littleton, Colorado, USA); Alexa Fluor 488 goat anti-mouse secondary antibody were from Molecular Probes (Eugene, Oregon USA). Mononuclear cells were prepared from buffy coats (Karolinska University Hospital Blood Bank, Stockholm, Sweden) obtained from healthy adult blood donors by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway) or Ficoll Paque (Amer- sham Pharmacia Biotech AB, Uppsala, Sweden), as described previously [38]. These studies were approved by the local ethical committee at Karolinska Institutet, Stockholm. Briefly, cells were washed and resuspended at 5.06106 cells/ml in RPMI-1640 medium. Monocytes were separated by adhesion to tissue culture plastic for 1 h at 37uC with a 5% CO2 atmosphere and non-adherent cells were removed by several washes with PBS. Human monocyte- derived macrophages (HMDMs) were cultured for 3–4 days in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco Invitrogen Corporation, Paisley, UK), and 50 ng/ml human recombinant M-CSF (50 ng/ml = 7500 IU/ml) (R&D Systems, Abingdon, UK). For isolation of dendritic cells, peripheral blood mononuclear cells were harvested as described above, and washed three times with PBS, followed by resuspension in MACS-buffer (80 ml/107 cells) containing 0.5% BSA, 2 mM EDTA in PBS. Anti- CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) were added according to the manufacturer’s instructions. After 30 min at 4uC, the CD14-positive cells were separated from the solution by autoMACS (Miltenyi Biotec), and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ) to check for CD14+ cell purity. Monocyte-derived dendritic cells (MDDC) were generated essentially as described before [39] by culturing monocytes in RPMI 1640 medium, supplemented with 25 mg/mL gentamicin, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 50 mM b-mercaptoethanol, 10% heat- inactivated FCS, and the recombinant cytokines GM-CSF (550 IU/ mL), and IL-4 (800 IU/mL), at a density of 4.06105 cells/ml, at 37uC in a 5% CO2 atmosphere. Both cytokines were from Biosource International (Camarillo, CA). After 6–7 days, the cell surface molecules CD1a, CD11c, CD14 and CD83 were analyzed by flow cytometry as described below, to confirm an immature, phagocytosis- competent phenotype with low CD83 expression. HeLa cells and RAW264.7 macrophages (American Tissue Culture Collections; ATCC) were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin. PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4398