Recent advances in nanotechnology have produced a multitude of nanoparticle types for use in many different products, including paints and inks, electroluminescent displays, solar panels, antimicrobials, cosmetics and sporting equipment. Semiconductor quantum dots (Qdots) are novel nanoparticles that have recently received a lot of attention because their unique photochemical and photophysical properties hold promise for a number of commercial applications including optoelectronics, counterfeiting inks, photovoltaics and biomedical imaging. Qdots have recently become broadly available to the scientific community through several commercial sources and many researchers are using Qdots as fluorescence imaging probes because their spectral properties are superior to traditional organic fluorophores [15, 16]. Qdots are several thousand times more stable against photobleaching than organic dye molecules and are thus well suited for continuous tracking studies over long time periods [17,18]. Qdots have large Stokes shifts and their broad absorption profiles allow simultaneous imaging of multiple colors within a biological sample and their emission wavelengths can be continuously tuned by varying particle size and/or chemical composition. Because of these unique optical properties, many of the current applications of Qdots have been focused on sensitive and quantitative bioimaging [15, 16,19], and on photosensitization and photoactivation therapies [20, 21]. In addition, we have recently demonstrated that Qdots can act as efficient intracellular delivery systems for novel therapeutics such as small inhibitory RNAs [22]. In general, the crystalline cores of Qdots are insoluble in water, and unless coated with a cap structure (e.g. ZnS) they also tend to be unstable, causing them to release their heavy metal core constituents making them less biocompatible and potentially toxic, thus limiting their usefulness for bioimaging and biomedical applications. In order to address the issues of water solubility, stability, biocompatibility, and toxicity, many groups have focused on the development of stabilizers and amphiphilic coatings to retain the photoluminescent properties of Qdots, while simultaneously providing a platform for further derivitization with antibodies, nucleic acids or other useful ligands [22-27]. We have recently synthesized CdSe/ZnS Qdots coated with tri-n-octylphosphine oxide (TOPO), and poly(maleic anhydride-alt-1-tetradecene (PMAT;a hydrocarbon polymer). These particles are exceptionally stable, retaining their fluorescence properties for at least 1 year when suspended in pH 7 phosphate buffered saline (PBS) and held at RT. Furthermore, the carboxyl functional groups present in this coating makes it convenient for ligand attachment. Similarly modified Qdots have shown promise for tumor targeting in vivo using antibodies directed against surface receptors on cancer cells [14, 28];and for siRNA delivery [22]. As indicated above, concerns have been expressed regarding the biocompatibility and toxicity of Qdots and this has been primarily focused on their potential to degrade in vivo, thus releasing cadmium, selenium or other toxic metals [24, 29, 30]. In addition, many studies of nanoparticle toxicity have focused on their ability to incite oxidative stress and/or free radical-based cellular injury [31-36], and some Qdots have been shown to cause oxidative stress in vivo in an invertebrate model [37]. Many nanoparticles are also known to be proinflammatory, especially when taken up by tissue macrophages that can elaborate pro-inflammatory cytokines and chemokines [38-40]. Furthermore, the surface modifications made to these particles may themselves be problematic, and might lead to undesirable effects such as thrombosis [41], or off target accumulation by reticulo-endothelial cells (primarily macrophages and dendritic cells) in the liver, spleen and lymph nodes [30, 40]. Several recent studies have addressed the potential of unstable Qdots or nucleic acid -modified Qdots to elicit a pro-inflammatory response in vitro and in vivo. Commercially available unstable non-capped Qdots were shown to be highly inflammatory when instilled into the lungs of ApoE null mice, an animal model of increased susceptibility to particle-induced lung injury [42]. These authors attributed the strong inflammatory response of these CdTe Qdots to the release of Cd. In another recent report, Qdots conjugated with the cDNA for enhanced green fluorescent protein (eGFP) were similarly shown to induce an inflammatory response in cultured mouse peritoneal macrophages in vitro, and in vivo after intraperitoneal injection in mice [43]. However, this study attributed the inflammatory response to the presence of nucleic acid on the surface of the Qdots because similarly constructed Qdots without the nucleic acid added were not inflammatory. In this investigation we established that TOPO-PMAT coated CdSe/ZnS Qdots are moderately toxic to cultured mouse and human macrophage-like cells, and that short-term exposure is associated with changes in the expression of proteins and cellular factors that are biomarkers of oxidative stress. Importantly, these Qdots also increased the expression and secretion of several pro-inflammatory cytokines, which suggests that they are likely to elicit an acute inflammatory response in vivo. Many particles and fibers are known to be proinflammatory, including crystalline silica, asbestos and ambient air particulates (e.g. diesel exhaust particles) which can have both local effects on the respiratory systems as well as more systemic effects. Nanoparticles such as carbon nanotubes, fullerenes and metal oxides (e.g. TiOa, CeOa) have also been shown to induce oxidative stress in vitro, and to cause inflammation in the lungs of exposed animals [44, 45]. In recent reviews of Qdot toxicity [29, 30], the adverse effects of Qdots were attributed to many factors, including Qdot instability and the release of the heavy metal core constituents, electronic structure, free radical generation, and in some cases the material used to cap the Qdot core (e.g. ZnS, mercaptoacetic acid, etc.). However, it is also recognized that in addition to the composition of the semiconductor core and capping materials, the constituents that are used to coat Qdots, and surface charge (zeta potential) are important determinants of cellular uptake and stability. In order to further stabilize Qdots and minimize their degradation, we synthesized them with a highly stable TOPO-PMAT polymer coating, which allows them to retain their fluorescent properties (an indication of core integrity) for extended periods of time. While the TOPO-PMAT Qdots were not found to be highly cytotoxic (likely due to their relative stability), we did note changes suggestive of an adaptive response to oxidative stress, including changes in NADPH and GSH levels. Because Qdots have been shown to induce oxidative stress in some models, we also decided to evaluate the expression of glutamate cysteine ligase (GCL) and heme oxygenase-1 (HMOX-1), two proteins whose expression is often induced when cells experience oxidative stress. GCL is the rate limiting enzyme in GSH synthesis and is composed of catalytic and modifier subunits (GCLC and GCLM, respectively). These GCL subunits are often induced in response to oxidative stress, primarily through the activation of the Nrf2/Keap1 pathway [46-48], and can thus serve as useful biomarkers of oxidative challenge. Increased levels of HM0X1 seemed to be the most consistent indicator of QD-induced stress measured in this study (Fig 7). HM0X1 catabolizes heme to biliverdin, which has antioxidant properties [49]. Induction of HMOX1 has been shown to be highly dependent upon the degradation of the transcriptional repressor Bachi [50], which can bind to antioxidant response elements (AREs) present in the promoters of many Phase II and oxidative stress-responsive genes ([51, 52]. Because Bachi has been shown to be degraded when cells are exposed to Cd, it may be that slow degradation of these Qdots in lysosomes releases enough Cd to effect this change in Bachi [53]. However, HM0X1 is.also responsive to a number of signals that increase the expression of pro-inflammatory cytokines, including NFkB [54]. It has recently been argued that HM0X1 is anti-inflammatory and may actually suppress the release of inflammatory cytokines [55], and thus HM0X1 induction may represent a means by which the inflammatory response and the oxidative stress associated with it are limited in nature. Our results suggest that TOPO-PMAT modified Qdots might elicit a pro-inflammatory response in vivo. The secreted cytokines we detected after exposure of both RAW and THP-1 cells to Qdots (IL-lb, TNF-a, MCP-1, MIP1-a, and MIP2) are important because they are known to be important for inflammation, and neutrophil and monocyte/macrophage influx into the mouse lung after particle injury [56-58]. Their expression is governed at least in part by NFkB-dependent transcription [59-62]. Other groups have also begun to examine the potential for various kinds of Qdots to cause inflammation. Hoshino and co-workers [63] reported on a study in which CdSe/ZnS Qdots were modified with a polycysteine/glutamine coating, conjugated with streptavidin, which were then used to bind biotinylated-eGFP cDNAs. Finally, biotinylated-nuclear localization signaling peptides (NLSP) were also bound to these Qdots to facilitate cellular uptake, nuclear localization and eGFP expression [63]. These Qdots were readily taken up by HEK293T cells, showed no/minimal toxicity, and were effective at delivering gene constructs, as evidenced by the expression of eGFP. However, in a subsequent and recently published paper, this same group assessed the ability of these eGFP cDNA-modified Qdots and albumin-biotin-streptavidin modified Qdots to cause inflammation in vitro and in vivo [A3]. The albumin-streptavidin modified Qdots repressed lymphocyte proliferation in vitro, but did not affect the ability of T cells to secrete IL-2 or IFNy after stimulated with anti-CD3 monoclonal antibody. Similariy, albumin-streptavidin modified Qdots did not significantly increase the secretion of the pro-inflammatory cytokines IL-6, IL-ip or TNFa by cultured mouse peritoneal macrophages. These authors also found no effect of these Qdots on inflammatory, homeostatic, or dual-function chemokines mRNAs in bone marrow derived or peritoneal macrophages exposed in vitro. However, when eGFP cDNAmodified Qdots were injected into the peritoneal space, there was a robust inflammatory response. Importantly there was no inflammatory response when the mice were injected with Qdots modified only with the NLSP, indicating that this inflammatory response was likely caused by the cDNA complexes (possibly by interacting with TLR9 on macrophages). Indeed, exposure of peritoneal macrophages to eGFP cDNA modified Qdots in v/Yro elicited a pro-inflammatory cytokine response (TNFa and MIP1a), whereas NLSP-modified Qdots did not. In a report by Jacobson and co-workers, the mRNAs for the inflammatory cytokines IL-6, MIP2 and MCP1, the % neutrophils and protein levels in BAL, and DNA damage in BAL cells were all found to be increased in the lungs of mice that had been exposed to CdTe Qdots by intratracheal instillation [42]. It should be noted that these Qdots contained no cap structure, and were only modified with either mercaptopropionic acid (negatively charged) or cysteamine (positively charged). Because these Qdots were presumably unstable, the authors attributed the strong inflammatory response to the release of Cd by this particular formulation of Qdots. In summary, even though the stable TOPO-PMAT modified Qdots used in this study were of limited toxicity, they did induce the release of pro-inflammatory cytokines/chemokines from cultured macrophage cell lines, suggesting that they may cause an inflammatory response in vivo. Preliminary data obtained with mice exposed to TOPO-PMAT Qdots via nasal instillation indicates that this is indeed the case (see Project 2). Many nanoparticle formulations are being developed as adjuvants for vaccines [40]. It may be that the proinflammatory responses seen in this study could represent an advantage in that it could increase the effectiveness of anti-tumor responses. Nonetheless, as with many new technologies, the excitement and promise of Qdots for medical therapies is tempered by the data we and others have collected regarding their potential to cause adverse effects.

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