Targeting Phagocytes by PS-CNT Figure 8. SWCNT functionalized with PS but not with PC engulfed in vivo by murine alveolar macrophages. A. a)Transmission electron micrographs of bronchoalveolar macrophages isolated from C57BL/6 mice exposed to PS-coated SWCNT; b) high magnification image of the area shown in (a). B. Alveolar macrophages preferentially phagocytoze PS-coated SWCNT in vivo. TEM images were used to calculate the percent of phagocytosis-positive macrophages. At least 150 cells were counted for each treatment. Data are mean6s.d., *p,0.05, PS-coated SWCNT vs non- coated or PC-coated SWCNT. C57BL/6 female mice, 7–8 weeks old, were exposed to SCWNT (40 mg/mouse) by pharyngeal aspiration. Twenty-four hours following exposure, mice were sacrificed using sodium pentobarbital. Mice were lavaged with sterile PBS. The lavage fractions were pooled and centrifuged to obtain the cellular fraction. Cells were then fixed for TEM. doi:10.1371/journal.pone.0004398.g008 provided more quantitatively reliable data. In this case, however, the limitations of quantitative assessments might be due to a possibility that fluorescently-labeled phospholipids could also modify (promote or decrease) the uptake of these fluorescent phospholipid coated SWCNT by macrophage. To minimize this interference, mole fraction of fluorescently-labeled phospholipids in the mixture with non-labeled phospholipids of the same type in all experiments did not exceed 10 mol%. Notably, the TEM-based evaluations of phagocytotic activity towards SWCNT were in good agreement with independent assessments performed by confocal imaging of NBD-labeled phospholipids-coated SWCNT. PS-induced responses initiate signaling cascades in macrophages, switching off their pro-inflammatory activation pattern, and turning on the production of anti-inflammatory cytokines and chemokines [15,73]. This suggests that functionalization in general, and PS- coating in particular, may change not only recognition but also pro-/anti-inflammatory behavior of macrophages interacting with nanoparticles. In line with this, our experiments demonstrated that PS coating of SWCNT could be utilized not only for directing SWCNT to phagocytic cells but also as a regulator or ‘‘switch’’ affecting the profile of the produced and released cytokines in vitro and in vivo [16,19]. Our studies show that PS-coating of SWCNT confers on them a ‘‘zip-code’’ address directing their recognition, engulfment, and uptake by professional phagocytes. Our results show that non-covalent attachment of cyt c to PS-coated SWCNTs, and subsequent release of cyt c inside macrophages using an endosome-disrupting agent, effectively activated caspase-3 in these cells, indicative of activation of apoptosis. These results are thus at variance with recent studies indicating that cellular uptake of functionalized nanotubes is independent of the nature of the functional group [9]. It must be noted, however, that the latter studies did not include professional phagocytes (macrophages) but rather a panel of non-phagocytic cell lines such as Jurkat, A549, HeLa cells, and so forth. Moreover, the studies by Kostarelos et al. [9] also indicated that the uptake in non-phagocytic cells could occur through passive penetration of the nanotubes through the plasma membrane. In contrast, one of the primary goals of our study was to develop approaches for targeting macrophages and other professional phagocytes by cytotoxic agents using nanotubes coated with PS as a specific ligand recognized by specialized plasma membrane receptors [25,26]. We chose to use cyt c as a ‘‘death’’ signal. Cyt c is one of the key co-factors for the activation of apoptosis in mammalian cells [62] and is known to be a difficult cargo for targeted delivery into cells [6,74], thus justifying the employment of cyt c as an appropriate ‘‘proof-of-principle’’ reagent. Our data show for the first time that PS-functionalized nanotubes could be used for selective delivery of specified cargoes (cyt c) into professional phagocytic cells, resulting in regulation of activity/ survival of these cells. Importantly, manipulating macrophage apoptosis can be a valuable therapeutic strategy in several diseases associated with the presence of intracellular pathogens in macrophages such as Mycobacterium tuberculosis and Listeria monocytogenes. Molloy et al. [75] showed that macrophage apoptosis resulted in reduced viability of intracellular mycobacteria. L. monocytogenes was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of macrophages which represent the predom- inant compartment of bacterial multiplication and die as a result of necrosis. Shifting the equilibrium from necrosis to apoptosis in L. monocytogenes infected macrophages is believed to constitute a promising therapeutic strategy [76]. Two other relevant examples are macrophage activation syndrome and rheumatoid arthritis - diseases where uncontrolled macrophage proliferation or macro- phage resistance to apoptosis, respectively, represents important features of disease pathogenesis [77,78]. Dai et al [79] have previously described the functionalization of SWCNT with a folate moiety for targeting of nanotubes to folate receptor-rich tumor cells in vitro, which could be considered for future cancer therapy. Mioskowski et al. [80] reported the self- assembly of several synthetic single-chain lipids around SWCNT to form supramolecular structures designed for the immobilization of histidine-tagged proteins; they did not investigate whether such nanotubes were ingested by cells. In addition, in a recent and elegant study, Liu et al. [81] reported on the attachment of an PLoS ONE | www.plosone.org 14 February 2009 | Volume 4 | Issue 2 | e4398