Specific interfacing of SWCNT with phagocytic cells of the immune system – macrophages, microglia, and dendritic cells - is important for several reasons. The first one is that SWCNT can be used for simultaneous targeted delivery of several different regulators/inhibitors with a potential to release them in temporally and spatially predetermined ways to control the bioactivity of a specific cell population during physiologically critical events. As macrophages can host a number of pathogens 
, nanocarriers can also be used for specific delivery of pro-apoptotic agents to aid in the defense against intracellular pathogens 
. Furthermore, macrophages and microglia are the major executors of pro-/anti-inflammatory responses, and - along with antigen-presenting dendritic cells - are important components of immune reactions 
. Specific targeting of cargoes/regulators to these cells could be exploited for therapeutic regulation of numerous immune functions, including the enhancement of immune responses to prophylactic or therapeutic allergen-specific vaccines through the coupling of allergens to nanocarriers. Finally, SWCNT are among the most commonly used nanomaterials with explosively expanding research and commercial applications 
. Because the production and employment of industrial quantities of SWCNT are becoming a reality, health risk concerns, particularly due to occupational and environmental exposures, are emerging 
. Not only an unusually large surface area, but also unique physical and chemical characteristics, redox features as well as significant decoration with transition metals alert to a possibility of unanticipated bioresponses resulting from interactions of SWCNT with cells, tissues, and biofluids. In fact, recent studies have demonstrated significant pulmonary and cardiovascular toxicity of SWCNT associated with a robust inflammatory response and early onset of fibrotic transition in mice 
. In this context, the enhancement of phagocytic recognition and uptake of SWCNT through PS-functionalization may be important in order to reduce the potential cytotoxicity of SWCNT.
Understanding of major principles of particle recognition by macrophages has long been a controversial issue. Because non-functionalized nanoparticles are prone to aggregation, sonication of non-coated SWCNT was performed before adding them to cells. Under these conditions, no significant uptake of non-coated SWCNT by RAW 264.7 macrophages occurred during the 2 h incubation period. In contrast, Dumortier et al. 
have recently reported that SWCNTs tend to re-aggregate and form large clusters that are eventually (after 24 h of co-incubation) phagocytozed by professional macrophages. It is likely that macrophage uptake of big clusters of SWCNT formed during prolonged incubation times is related to the reduced solubility of these non-coated nanotubes. It has been also reported that geometry and shape act as determinants of particle recognition 
. Several studies have demonstrated that functionalized SWCNT are recognizable by cells and taken up through endocytosis-dependent pathways 
. By contrast, we and others reported that non-functionalized SWCNT are neither effectively recognized nor phagocytozed by macrophages 
. The fact that PS-coating leads to recognition and uptake of SWCNT suggests that it is the lack of a recognition signal that is responsible for poor uptake of non-functionalized carbon nanotubes by phagocytes. A variety of specialized receptors on the macrophage surface have been implicated in recognition and tethering of different particles, including ultra-fine particles 
. Recent studies identified several novel macrophage receptors for which PS is a specific high-affinity ligand 
, facilitating uptake of target cells with externalized PS. PC is not specifically recognized by these receptors and its presence on the cell surface does not enhance recognition and uptake of cells by macrophages. Therefore, we chose to use NBD-PC-coated SWCNT as controls in our comparative experiments on assessments of SWCNT uptake by macrophages. Further, we utilized Annexin V – a protein known to selectively bind to PS (but not to PC) and mask PS recognition and uptake by macrophages. The Annexin V mediated suppression of uptake of NBD-PS-coated SWCNT (and lack of fluorescence response from macrophages incubated in the presence of NBD-PS coated SWCNT pre-treated with Annexin V) was employed as an additional specific control of PS-dependent recognition and uptake. Our data indicate that eclipsing of PS with its specific ligand, Annexin V, completely blocks SWCNT recognition by macrophage cell lines and primary phagocytes. Most notably, these PS-dependent recognition patterns are realized in vivo
whereby alveolar macrophages display enhanced uptake of PS-coated SWCNT during an inflammatory pulmonary response induced by aspiration exposure. Together, our in vitro
and in vivo
studies demonstrate that PS-functionalization renders nanotubes appetizing to phagocytes. We believe that analysis of SWCNT uptake by phagocytes based on the employment of fluorescently-labeled phospholipids (NBD-PS and NBD-PC) 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 
. 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 
. 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 
. 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. 
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 
. 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 
and is known to be a difficult cargo for targeted delivery into cells 
, 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. 
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 predominant 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 
. Two other relevant examples are macrophage activation syndrome and rheumatoid arthritis - diseases where uncontrolled macrophage proliferation or macrophage resistance to apoptosis, respectively, represents important features of disease pathogenesis 
Dai et al 
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. 
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. 
reported on the attachment of an 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 
) 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 
. 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 populations. 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 macrophages (eg. superoxide-sensitive linkers) or intracellularly (eg. esterase-sensitive conjugates) for targeted delivery and release to regulate life-span and activity of phagocytes.