phospholipid, phosphatidylserine (PS), which is normally confined to the cytosolic leaflet of plasma membrane [17,18]. Thus, externalization of PS during apoptosis generates an ‘‘eat-me’’ signal for macrophages. Notably, non-apoptotic cells with externalized PS can also be taken up by macrophages and suppress ROS and RNS production [19]. PS externalization is one of several features contributing to recognition of apoptotic cells. Chemotactic factors such as lyso-PC [20], and bridging molecules such as MFG-E8 [21] are some examples of other important participants in the process of apoptotic cell clearance. Moreover, additional recognition signals on the surface of apoptotic cells have also been shown to be involved in this process, including proteins such as annexin I and calreticulin [22,23]. However, PS remains a universal component of the recognition pattern on the surface of apoptotic cells [24], and recent studies have implicated several different macrophage receptors in the process of PS-dependent clearance of cell corpses [25,26]. In addition, knockout-mouse studies have shown that PS-dependent clearance of apoptotic cells is crucial for the maintenance of tissue homeostasis [27]. Therefore, we hypothesized that coating of SWCNT with PS will interface them with macrophages and stimulate the recognition, tethering and engulfment of nanotubes. Thus, PS-coated SWCNT can be utilized for targeted delivery of specialized cargos - regulators, inhibitors - into macrophages to control their functions including inflammatory responses to SWCNT themselves. Here we report that PS-coated SWCNTs are indeed readily taken up by various classes of phagocytic cells (macrophages, microglia, and dendritic cells). Further, using PS-coated SWCNTs we were able to successfully deliver cytochrome c (cyt c), a pro-apoptotic death signal – and cause apoptosis in macrophages. These studies thus demonstrate that non-covalent modification of SWCNTs with specific phospholipid molecules can be employed for targeted delivery and regulation of professional phagocytes. Materials and Methods Reagents 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), 1,2-Dio- leoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (DOPS), 16:0-6:0 NBD PS, 1-Palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadia- zol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phospho-L-Serine (Am- monium Salt) and 16:0-6:0 NBD PC, 1-Palmitoyl-2-[6-[(7-nitro-2- 1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phospho- choline (Ammonium Salt) were from Avanti Polar Lipids Inc. (Alabaster, AL). HEPES, MgCl2, KCl, NaCl, phenylmethylsulfonyl fluoride, glutaraldehyde, osmium tetroxide, potassium ferricyanide, diethylenetriaminepentaacetic acid (DTPA), zymosan and Hoechst 33342 were from Sigma-Aldrich (St. Louis, MO). RPMI, DMEM medium, Ca2++Mg2+-free PBS were purchased from Invitrogen Corporation (Grand Island, NY). Particles SWCNT (CNI Inc., Houston, TX) produced by the high pressure CO disproportionation process (HiPco) technique [28], employing CO in a continuous-flow gas phase as the carbon feedstock and Fe(CO)5 as the iron-containing catalyst precursor, and purified by acid treatment to remove metal contaminates [29] were used in the study. Chemical analysis trace metal (iron) in SWCNT was performed at the Chemical Exposure and Monitoring Branch (DART/NIOSH, Cincinnati, OH) using nitric acid dissolution and inductively coupled plasma-atomic emission spectrometry (ICP-AES). Analysis revealed that SWCNT comprised of 0.23 weight % iron. For purity assessment of Hipco SWCNT, we used several standard analytical techniques including Targeting Phagocytes by PS-CNT thermo gravimetric analysis with differential scanning calorimetry (TGA-DSC), thermo-programming oxidation (TPO), and Raman and Near-Infrared (NIR) spectroscopy [30]. Comparative analyt- ical data obtained by TGA-DSC, TPO, NIR and Raman spectroscopy revealed that more than 99% of carbon content in the SWCNT HiPco product was accountable in CNT morphol- ogy. Purified suspended HiPco SWCNT [29] were used in the study. SWCNT were routinely tested for bacterial endotoxin (LPS) contamination using the endpoint chromogenic LAL method, as previously described [31]. The mean diameter and surface area of SWCNT was 1–4 nm and 1040 m2/g. Surface area was determined by Brunauer, Emmett, and Teller (BET) analysis, and diameter and length was measured by TEM. The chemical cutting of SWCNT was performed as reported previously [32]. Purified SWCNT were dispersed in 4:1 mixture of concentrated H2SO4 and 35% aqueous H2O2 and sonicated in ultrasonic bath (Branson 1510 SonifierH, output power of 70 W at 40 KHz) for 24 hrs at 0uC. The dispersion was then heated to 70uC for 10 min for ‘‘polishing’’ the nanotubes. This solution was then diluted 10-fold by deionized water and filtered through PTFE membrane (100 mm pore size). The collected sample was thoroughly washed with deionized water and vacuum dried at 110uC for 30 min. Thus obtained short SWCNT were dispersed in 25 mM HEPES buffer (pH 7.4; containing 150 mM NaCl) by sonication to final concentration 0.5 mg SWCNT/ml. Transmission electron microscopy was conducted in a FEI- Morgani TEM operated at 80 KV equipped with a soft imaging system charge-coupled device (CCD) camera. TEM samples were prepared by drop casting the solution on copper grid and the excess drawn off with filter paper. The grid was negatively stained with 2% uranyl acetate solution for a minute. Zeta potential and particle size were determined on the Malvern Zetasizer Nano (Malvern Instruments, Westborough, MA). The analysis was conducted according to standard operating procedure (Particle Technology Labs, Ltd, Downers Grove, IL) for this instrumentation type. Elemental carbon was analyzed using modified method NIOSH 5040 (NMAM 5040) [33]. After sonication and vortexing, samples (50 ml) were spiked onto 1.45 cm2, clean quartz filter punch and placed in Petri dish. The samples were allowed to dry overnight in a dessicator located in the balance room. The quartz punch was then analyzed with the Sunset thermal-optical carbon analyzer (Sunset Laboratory Inc., Tigard, OR). Atomic force microscopy (AFM) After sonication, 20 mL of SWCNT in 25 mM HEPES buffer (pH 7.4) was placed on a freshly cleaved mica which was subsequently rinsed with DI water while being spun at 950 rpm to remove the excess of the buffer solution. The samples were air- dried prior to imaging. AFM images were collected in a tapping mode with a Multimode Nanoscope IIIa microscope (Digital Instruments, Santa Barbara, CA) in air. Coating of SWCNT with phospholipids and other cargoes In all presented experiments, the SWCNT subjected to chemical cutting by H2SO4 plus H2O2 have been utilized. The morphology of thus obtained SWCNT preparations was assessed by TEM, SEM as well as AFM. Following the chemical cutting of SWCNT, the nanotubes were used for coating with cargoes (phospholipids, cyt c). Therefore, neither phospholipids nor cyt c were exposed to aggressive environments employed for the SWCNT cutting protocol. SWCNT were sonicated with either 2.5 mM DOPC or 5 mM DOPC: DOPS at the ratio of 1:1 (3 cycles 30 s), then washed 4 times with 25 mM HEPES, pH 7.4. PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4398