Transmission Electron Microscopy Macrophages were fixed in 2.5% glutaraldehyde for 1 h, pelleted, and re-suspended in 3% gelatin in PBS, olidified at 4uC, and then re-fixed for 15 min. Pellets were washed 3 times in PBS and then postfixed in 1% OsO4 and 1% K3Fe(CN)6 for 1 hour. After 3 PBS washes, the pellet was dehydrated through a graded series of 30% to 100% ethanol then incubated in Polybed 812 epoxy resin (Polysciences, Warrington, PA) for 1 h. After several changes of 100% resin over 24 h, pellet was cured at 37uC overnight with additional hardening at 65uC for 48 h. Ultrathin (60 nm) sections were collected on 200 mesh grids and stained with 2% uranyl acetate in 50% methanol for 10 min followed by 1% lead citrate for 7 min. Sections were observed on a JEM 1210 electron microscope (JEOL, Peabody, MA) at 80 kV. Statistics The results are presented as mean6s.d. values from at least three experiments, and statistical analyses were performed by either paired/unpaired Student’s t-test or one-way ANOVA. The statistical significance of differences was set at p, 0.05. Results Physico-chemical characterization of functionalized SWCNT We prepared SWCNT coated with DOPC, as a control (PC- coated SWCNT), or a mixture of DOPS, plus PC (PS-coated SWCNT) by incubating nanotubes in the presence of liposomes containing these lipids (PC or a mixture of PS plus PC at a molar ratio of 1:1). Successful integration and the content of PS and PC associated with SWCNT were confirmed by direct assessment of the phosphorus content after HPTLC separation of phospholipids (Fig. 1Aa). We found that PC-coated SWCNT contained 462633 nmol PC/mg SWCNT. Analysis of PS-coated SWCNT revealed that amounts of integrated PS and PC on nanotubes were approximately similar: 198614 and 223613 nmol of phospho- lipids/mg SWCNT, respectively (Fig. 1Ab). Additionally, the presence of PS on the surface of SWCNT was verified using FITC-conjugated PS-specific protein, Annexin V, by measuring its characteristic fluorescence (Fig 1B). After treatment of PS-coated SWCNT with FITC-conjugated Annexin V, the appearance of a robust fluorescence response was observed. Analysis performed by NMAN 5040 revealed that purified SWCNT were comprised of 99.7 wt% elemental carbon. Determinations of carbon content for non-coated, PS-coated, PC-coated and cyt c/PS-coated SWCNT showed that non- covalent functionalization of SWCNT with phospholipids and protein (cyt c) expectedly resulted in increased carbon content in SWCNT suspensions. Based on our HPTLC-based determina- tions of phospholipid content of PC- and PS-coated SWCNT, the expected content of elemental carbon in the suspensions of these coated SWCNT should be 81 and 61 ng/ml, respectively. Direct estimates of elemental carbon content were 75 and 59 ng/ml for SWCNT functionalized with PS and PC (Table S1). These numbers correspond to 87% and 97% of theoretically calculated content of the two phospholipids, respectively. There was essentially no difference in the organization and structure of SWCNT between non-coated samples and PS- or PC- coated samples. Assessments of size distribution by dynamic light scattering (DLS) showed that SWCNT (Fig. 1Ca), PS-coated SWCNT and PC-coated SWCNT samples in suspensions had very similar particle sizes in the range between 130–180 nm (Table S1). Thus, SWCNTs were similarly dispersed in all formulations used. Statistical analysis of isolated SWCNTs Targeting Phagocytes by PS-CNT measured by TEM revealed length distribution with a mean length of 90635 nm (Fig. 1Cb). DLS determined the average hydrodynamic diameter of the same sample as <135 nm. Average particle sizes from DLS measurements for all samples are summarized in Table S1. Small increases in particle size indicate that no significant aggregation of SWCNTs takes place in solution. Moreover, it is known that in microscopy, assessments may be length-biased because they do not include nanotubes that cross the edge of the micrograph and those that aggregate [41]. In fact, longer nanotubes are ignored, because they are more likely to reach out of view and aggregate with each other. Evaluations of zeta potentials for SWCNT, PC-coated SWCNT, PS-coated SWCNT, PS-NBD-coated SWCNT, PC- NBD-coated SWCNT, and SWCNT containing cyt c showed that they ranged from 240 to 250 mV (Supplemental Table 1). Coating of SWCNT with phospholipids caused a slight increase in the negative values of zeta potentials which, however, was not significant between PC-coated SWCNT and PS-coated SWCNT. Overall, the estimated negative values of zeta potentials corre- spond with significant stability of dispersed non-coated and phospholipids-coated SWCNTs in aqueous suspensions [42,43]. Transmission electron microscopy (TEM) of negatively stained non-coated, PS-coated, and PC-coated SWCNT showed that they had typical fibrous morphology and were represented mostly by single nanotubes, as well as by ropes of nanotubes (Fig. 1Cb) and entangled ‘‘bird’s nest’’-like aggregates. (Figure S1A). Calculated percentage contributions of each of these ‘‘morphologies’’ in the formulations used were 70, 25 and 5% for single SWCNT, bundles and ‘‘bird’s-nest’’-like aggregates, respectively (Fig 1Cb insert). PS-coated and PC-coated SWCNT had similar morphol- ogy as non-coated SWCNT. Moreover, a thin layer of phospholipids (PS/PC) (stained) is clearly visible on the sidewalls of SWCNT by TEM (Figure S1B). Cytotoxicity of SWCNT Both Trypan Blue exclusion test and LDH release assay demonstrated that neither non-coated SWCNT nor phospholip- ids-coated SWCNT induced any cytotoxic effects in RAW 264.7 macrophages after 4 hrs of co-incubation (Figure S2). Moreover, no impairment of viability was seen at 4 and 24 h of incubation of SWCNT with primary human macrophages, using the Trypan blue assay and the Hoechst 33342 staining method for visualization of cell nuclei (data not shown). Scanning electron microscopy (SEM) imaging of SWCNT To investigate interactions of SWCNT with the surface of macrophages, we performed SEM imaging (Fig. 2A). We found that PS-coated SWCNT were actively recognized and tethered to the surface of RAW264.7 macrophages. At higher magnifications (Fig. 2Aa), we were able to detect the presence of SWCNT fibers on the surface of macrophages. Numerous filopodia – budding off the macrophages surface – associated with their major functions such as phagocytosis and substrate adhesion were observed in RAW264.7 macrophages exposed to PS-coated SWCNT. In contrast, non-coated (Fig. 2Ab), PC-coated (Fig. 2Ac) and PS- coated Annexin V treated (Fig. 2Ad) SWCNT were ingested to a lesser extent by macrophages, and the presence of these types of SWCNT on the surface of RAW264.7 macrophages was observed much less frequently. Transmission electron microscopy (TEM) of SWCNT TEM evaluations of the uptake of SWCNT showed that RAW264.7 macrophages readily phagocytozed PS-coated SWCNT (Fig. 2Ba) while a significantly less pronounced PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4398