Targeting Phagocytes by PS-CNT Figure 5. Primary rat microglia recognized SWCNT functionalized with PS but not with PC. A. Scanning electron micrographs of microglia treated with PS-coated SWCNT in vitro. Microglia (1.56105 cells/ml) were incubated for 2 h with PC- or PS-coated SWCNT. At the end of incubation macrophages were washed and fixed for SEM. (Note: The red arrows in all the electron micrographs in figure 5 point SWCNT). B. Transmission electron micrographs of primary microglia exposed to SWCNT in vitro. Microglia (1.56105 cells/ml) were incubated with PC- or PS- coated SWCNT. At the end of incubation microglia was washed and fixed for TEM. a) Microglia exposed to PS-coated SWCNT; b) Quantitative assessments of SWCNT phagocytosis by RAW 264.7 macrophages. A total of 60 cells from each sample type were analyzed by TEM. Data are mean6S.D., n = 3, *p,0.05, PS-coated SWCNT vs SWCNT, PC coated SWCNT and PS-coated Annexin V–treated SWCNT. C. In vitro assessment of uptake of SWCNT coated with NBD-PS or NBD-PC by microglia. Microglia (1.56105 cells/ml) were incubated with NBD-PC- (a), NBD-PS-coated SWCNT (b) or NBD-PS-coated/Annexin V treated SWCNT (c). 1 – bright field image; 2 – blue fluorescence image, Hoechst 33342; 3 – green fluorescence image, NBD-labeled phospholipids; 4 – overlap of blue and green fluorescence images with image under bright field. d) Annexin V treatment of NBD-PS- coated SWCNT prevents their engulfment by microglia. Quantitative evaluation of cell number with engulfed SWCNT. Data are mean6s.d., n = 4. *p,0.05, NBD-PS-coated SWCNT vs NBD-PC-coated SWCNT and NBD-PS-coated Annexin V treated SWCNT. doi:10.1371/journal.pone.0004398.g005 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 [54,55], nanocar- riers can also be used for specific delivery of pro-apoptotic agents to aid in the defense against intracellular pathogens [56]. 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 [57–60]. 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 [61]. 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 [62]. 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 [53,63,64]. 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. PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4398