RGD peptide to nanotubes coated with polyethylene glycol (PEG) and subsequent in vivo administration to mice bearing integrin- positive tumors. Efficient targeting of the nanotubes to the tumors was achieved through this approach. The goal of our work was to employ the naturally occurring ‘‘eat-me’’ signal, PS as a specific ligand that is recognized by professional phagocytes. We provide evidence that the uptake is specific, since nanotubes functionalized with PC are not recognized as readily as PS-coated nanotubes and because the PS-binding protein, Annexin V, can prevent uptake of PS-coated nanotubes. Because the chemical cutting is associated with the production of oxidative negatively-charged defects in SWCNT (carboxy-groups [32]) it is possible that coating with phospholipids could be slightly different in precut and non-precut SWCNT. It should be noted, however, that binding of negatively charged PS (at pH 7.4) is unlikely to occur at the sites of negatively charged defects due to electrostatic repulsions. Previous studies from our and other laboratories have established high-affinity binding of cyt c with PS [82]. After the coating, binding of positively-charged cyt c was likely associated with abundant negatively-charged PS. We also provide evidence that the mechanism of uptake is specific and occurs through endocytosis. Finally, we show that a relevant cargo (pro-apoptotic cyt c) can be delivered into macrophages, and upon disruption of endosomes, the cargo is released and can perform its biological function (activation of the caspase cascade). Taken together, our findings highlight a novel strategy for the controlled delivery of relevant cargoes into specific cell popula- tions. Further developments of the novel principle established in the current study, i.e. PS-coating of SWCNT for recognition and ingestion, may exploit cargoes covalently-conjugated with specific linkers that are hydrolysable extracellularly by activated macro- phages (eg. superoxide-sensitive linkers) or intracellularly (eg. esterase-sensitive conjugates) for targeted delivery and release to regulate life-span and activity of phagocytes. Supporting Information Figure S1 TEM images of bare SWCNT (A) and PS-coated SWCNT (B) deposited on mica substrates. Found at: doi:10.1371/journal.pone.0004398.s001 (5.93 MB TIF) Figure S2 Viability of RAW264.7 macrophages exposed to SWCNT. RAW 264.7 macrophages were seeded at 16104 cells/ well on 96 well-plate 12 hrs before treatment. Cells were incubated with non-coated SWCNT or SWCNT coated with either PS or PC in serum and phenol red free DMEM containing References 1. Gonzalez-Flecha B (2004) Oxidant mechanisms in response to ambient air particles. Mol Aspects Med 25: 169–182. 2. Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, et al. (2004) Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 16: 437–445. 3. Elder A, Oberdorster G (2006) Translocation and effects of ultrafine particles outside of the lung. Clin Occup Environ Med 5: 785–796. 4. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, et al. (2005) Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol 289: L698–708. 5. Kagan VE, Tyurina YY, Tyurin VA, Konduru NV, Potapovich AI, et al. (2006) Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron. Toxicol Lett 165: 88–100. 6. Kam NW, Dai H (2005) Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J Am Chem Soc 127: 6021–6026. 7. Nimmagadda A, Thurston K, Nollert MU, McFetridge PS (2006) Chemical modification of SWNT alters in vitro cell-SWNT interactions. J Biomed Mater Res A 76: 614–625. 8. Klumpp C, Kostarelos K, Prato M, Bianco A (2006) Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics. Biochim Biophys Acta 1758: 404–412. Targeting Phagocytes by PS-CNT 100 mM DTPA for up to 4 hrs. At the end of incubation, the culture medium was collected, centrifuged to remove SWCNT and used for LDH assay with CytotoxOne kit. Data are mean6s.d., n = 3. Found at: doi:10.1371/journal.pone.0004398.s002 (0.50 MB TIF) Figure S3 PS-coated SWCNT effectively deliver cyt c into RAW264.7 macrophages, and activate apoptotic pathways (caspase 3/7). RAW264.7 macrophages were seeded at 0.56105 cells/well 12 hrs prior to treatment. Cells were incubated with differently functionalized SWCNT in serum and phenol red free DMEM containing 100 mM DTPA for 1 hr at 37uC. At the end of incubation, cell were washed to remove SWCNT and treated with chloroquine (100 mM) in complete culture medium at 37 uC for 15 min. After that, cells were washed with complete DMEM and additionally incubated for 8 hrs. Cells were stained in binding buffer containing annexin V (0.5 mg/ml) for 5 min and counter- stained with Hoechst 33342 for nuclei at room temperature. The cells were examined under a Nikon ECLIPSE TE 200 fluorescence microscope (Tokyo, Japan) equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR) and analyzed using the MetaImaging SeriesTM software version 4.6 (Universal Imaging Corp., Downingtown, PA). A minimum of 300 macrophages were counted per experimental condition. Data are mean6s.d. (* p,0.01 vs untreated). Insert: Typical fluores- cence image of RAW264.7 macrophages treated with cyt c/PS- coated SWCNT (staining with annexin V). Found at: doi:10.1371/journal.pone.0004398.s003 (1.39 MB TIF) Table S1 Found at: doi:10.1371/journal.pone.0004398.s004 (0.03 DOC) MB Acknowledgments DISCLAIMERS: The findings and conclusions in this report are those of the authors and do not necessarily represent the view of the National Institute for Occupational Safety and Health. Author Contributions Conceived and designed the experiments: NVK YYT WF LVB NAB HB KC DS HV AS EW BF PDK AS ERK ARM AAS VEK. Performed the experiments: NVK YYT WF LVB NAB HB KC MR DS HV AS EW BF PDK ERK ARM AAS. Analyzed the data: NVK YYT WF LVB NAB HB KC MR HV AS EW BF PDK AS ERK ARM AAS VEK. Contributed reagents/materials/analysis tools: DS BF AS AAS. Wrote the paper: NVK VEK. 9. Kostarelos K, Lacerda L, Pastorin G, Wu W, Wieckowski S, et al. (2007) Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nature Nanotechnology 2: 108–113. 10. Chen X, Tam UC, Czlapinski JL, Lee GS, Rabuka D, et al. 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Huynh ML, Fadok VA, Henson PM (2002) Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation. J Clin Invest 109: 41–50. PLoS ONE | www.plosone.org 15 February 2009 | Volume 4 | Issue 2 | e4398