QD are being developed and used for a variety of applications (e.g., drug delivery, diagnostic procedures) that may lead to human exposure in medical settings. In addition, the number of workers producing or using nanomaterials, such as QD, is growing substantially. Unfortunately, little information exists about the potential health hazards associated with medical, environmental, or occupational lung exposures to QD. One important occupational health issue that is being addressed by the National Institute for Occupational Safety and Health (NIOSH) Nanotechnology Research Center is determining the toxic potential and fate of nanomaterials in the body after exposure . Therefore, there is interest in assessing the potential adverse health outcomes that may occur following pulmonary QD exposure, and the mechanisms underlying these responses. NIOSH is actively performing field studies to evaluate the potential exposure of workers in the QD industry (personal communication with Kenneth Martinez, 2012).
In vivo animal studies assessing the pulmonary responses after exposure to QD are limited. The QD samples examined in the current study were composed of a CdSe core. Cd is a widespread environmental and occupational pollutant and has been classified as a type I carcinogen by the International Agency for Cancer Research (IARC ). Inhalation of Cd-containing pollutants can result in metal fume fever, chemical pneumonitis, pulmonary edema, and pleural effusion . In addition to composition and the presence of potentially toxic metals, surface coating, charge, and size may influence QD toxicity. In vivo lung toxicology studies have shown that nanoparticles of identical composition and mass may be more toxic (Brown et al. [16, 17]) when compared to their larger counterparts. In vitro studies indicate that Cd-containing QD with a smaller diameter had greater toxicity and distributed to the nucleus of the cell compared to a larger QD of the same composition that were less cytotoxic and remained in the cell cytoplasm . Functionality and surface charge of QD are also of important in regard to conjugation to other particles or molecules for targeted delivery of substances in the body or tracking of substances. The QD in the current study were functionalized with different surface moieties by the manufacturer to examine the effect of surface charge on toxicity and were determined to be in the ultrafine size range with core size diameters of 5–10 nm.
A significant increase in lung injury was observed at different time points after a single intratracheal instillation of the QD-COOH and QD-NH2 samples in the current study. Lung injury parameters, LDH and albumin, were significantly increased immediately after treatment with the 12.5 μg dose, peaked at day 7 post-exposure, and remained elevated throughout the 28 day period, even though the response at 28 days was subsiding. Evidence for lung injury was not apparent until 5 days after treatment with the 5 μg dose of QD-NH2 sample where it was found to be more potent than the QD-COOH treatment. At 7 days post-exposure, the 5 μg dose of the QD-COOH sample began to exhibit toxicity and was more potent than the QD-NH2 sample. The difference in temporal response between the two doses may be related to the lungs responding earlier to a substantially greater number of particles at the higher 12.5 μg dose (e.g., dose effect), whereas the later injury response after treatment with the 5.0 μg dose may better reflect a potential mechanism of toxicity, the in situ dissolution of the QD by AM, leading to the direct exposure of the lung cells to free Cd (e.g., chemical effect). Moreover, the QD-NH2 sample induced significantly more lung injury at earlier time points after treatment compared to the QD-COOH, which may be due to a difference in particle distribution related to charge or a slightly faster disruption of the QD-NH2 structure, allowing for cellular exposure to free Cd sooner, or a combination of the two. Multiple studies have indicated that coating affects destabilization of QD and that uncoated Cd-containing QD released free Cd from the QD core intracellularly and were cytotoxic in vitro
[19, 20]. In addition, Derfus et al.  demonstrated that the encapsulation of Cd/Se QD by a ZnS shell greatly reduced both the amount of free Cd released and the production of free radicals by QD.
In terms of lung inflammation as determined by the number of PMN recovered from the lungs, a significant inflammatory response was observed at 1 day after treatment with the 12.5 μg dose, likely due to a dose/bolus effect because this initial response subsided by 3 days after treatment, before increasing again and peaking at day 7. In addition, the lower dose QD treatment groups had no effect on inflammation at 1, 3, and 5 days after treatment. As was seen with the lung injury response, the number of recovered lung PMN was significantly greater at earlier time points (days 3 and 5) after treatment 12.5 μg dose of QD-NH2 sample compared to the QD-COOH sample, whereas PMN influx was greater in the QD-COOH-treated rats at days 14 and 28 when compared to those treated with QD-NH2, again implicating a likely difference in the dissolution rate between the two QD samples and the release of free Cd. Despite these differences, the pattern of inflammation is similar between QD groups, with peak responses between 7–14 days after treatment for the 5 and 12.5 μg dose groups for both QD samples. In addition to PMN influx, AM infiltration peaked at these two time points as well. In agreement with the inflammatory response seen in the current study, Ma-Hock et al.  also observed a transient local PMN inflammation in the lungs of rats that was associated with increases in inflammatory chemokines, MCP-1 and MIP-2, after a short-term inhalation of Cd-containing QD (cadmium sulfide core with a cadmium hydroxyl shell – CdS/Cd(OH)2)). This delay in inflammatory cell influx into the lungs after treatment is additional evidence that free Cd released from the dissolution of the QD structure is likely a causative factor responsible for the pulmonary responses observed. The lung inflammation pattern was similar to that of lung injury and appeared to be transient with the response subsiding (even at the highest dose) by 28 days after treatment with both QD samples.
Regarding lung deposition and clearance, QD-COOH and QD-NH2 were both rapidly phagocytized in vivo by AM in a dose-dependent manner as early as 2 hr after intratracheal instillation. Cd burden in LALN beginning 1 week after exposure suggests that at least a portion of the QD in the alveolar region are cleared via scavenging phagocytes which then migrate out of the lung through the lymphatics. Ma-Hock et al.  also observed AM clearance to the LALN as well. Translocation from the lung and clearance via the blood is also a possible route of clearance for particles in the lung. Confocal microscopy revealed some QD to have deposited on the epithelium outside AM and move to the interstitium, giving this portion of QD the potential of reaching the systemic circulation and translocating to other organs. However, measureable levels of Cd as determined by neutron activation did not appear in a systemic organ until 7 and 14 days after treatment with the QD-COOH and QD-NH2 samples, respectively. Using an in vitro model, Geys et al.  examined the effect of surface charge on the translocation of non-functionalized QD, QD-COOH, and QD-NH2 through a tight monolayer of primary rat alveolar epithelial cells. They observed that QD did not translocate through the tight junctions of alveolar epithelial cells, regardless of surface charge, without disruption of the cell-to-cell barrier induced by oxidative stress. In the current study, Cd depositing in extrapulmonary organ systems corresponds to the time points post-exposure where the greatest lung injury is observed, rendering it likely that the translocation of QD is occurring, and that this may be a passive process via “leaky” cell junctions, rather than an active process.
Neutron activation showed that at day 1 there was more Cd measured in the lungs of rats treated with QD-COOH when compared to those treated with QD-NH2. Uneven distribution due to route of administration (intratracheal instillation resulting in more QD going to one bronchus or the other), differences in deposition pattern (greater deposition in upper airways versus parenchyma), or differences in destabilization and translocation of the particles in the first 24 hours may contribute to this discrepancy. Differences in the rate of clearance were evident with greater percentage of QD cleared from the lungs by day 28 in the QD-COOH group compared to the QD-NH2 group, relative to early deposition amounts. However, the pattern of clearance via the lymphatics, and translocation to the kidneys via the blood stream, were similar between the two QD, and neutron activation analysis showed that Cd persisted in the lungs at least 28 days after exposure for both QD. This data is in agreement with other studies. Using inductively coupled plasma optical emission spectroscopy (ICP-OES) as opposed to neutron activation, Ma-Hock et al.  also observed that Cd levels in the lungs did not decline during a 3-week recovery period after inhalation exposure to water soluble CdS/Cd(OH)2.
Silver enhancement staining of lung tissue recovered from the QD-treated animals in the current study provided additional evidence that Cd persisted in the lungs at 28 days after treatment. The silver staining, used to image deposited metal particles, taken into consideration with the diminished fluorescent signal observed 1 week after exposure to QD using confocal microscopy, is another indication that free Cd had dissociated from the QD beginning 5–7 days after treatment. Although both forms of QD persisted in the lung up to 28 days, the rate of clearance appears to differ between the positively and negatively charged particles, with a slower clearance rate observed for QD-NH2 particles. This difference in the clearance patterns may be due to different dissolution rates of Cd from the QD after AM phagocytosis and enzymatic digestion because of the different surface charges and properties of the two QD samples as discussed above. In addition, a greater influx of phagocytes was observed at the later time points post-exposure to QD-COOH when compared to QD-NH2, resulting in a greater capacity for clearance. Also, it should be noted that initial Cd deposition in the lungs was greater with the QD-COOH sample compared to the QD-NH2 sample and this may contribute to a greater initial clearance of QD-COOH. As mentioned above, for both QD, a portion of the clearance appeared to occur via mechanical clearance by AM, as well as by extrapulmonary translocation.
The kidney, a primary target organ of Cd toxicity, was the only extrapulmonary organ in which Cd was measured in the current study. Relatively small concentrations of Cd appeared in the kidneys at 7 days after QD-COOH treatment and at 14 days after QD-NH2 treatment with levels increasing at 28 days for both groups. Ma-Hock et al.  also observed small amounts of Cd in kidney, which increased very slightly over the post-exposure recovery period. Importantly, histological analysis of kidney in their study did not indicate any morphological abnormalities after short-term QD inhalation. The Cd levels measured in the kidney of the current study were substantially lower than the levels of 104–252 μg/g wet kidney that were observed to cause renal dysfunction in rats after oral Cd treatment . The presence of Cd in the kidney is in line with the findings of Chen et al.  and Lin et al.  who observed the accumulation of QD in the kidney of rats and mice, respectively, after intravenous injection. However, Ma-Hock et al.  measured Cd in the liver after inhalation, and both Chen et al.  and Lin et al.  observed QD in the liver after intravenous exposure. Measureable levels of Cd were not detected in the liver after intratracheal treatment of the different QD samples in the current study, likely due to dose and route of administration combined. In agreement with the Ma-Hock et al.  study, lung treatment did not lead to the accumulation of Cd in the brain or spleen in the current study.
Based on the findings of the current study and others, several conclusions can be made concerning the toxicity and fate of QD after pulmonary treatment. Lung exposure to high enough concentrations of CdSe QD can cause transient lung inflammation and injury that appears to be related to the dissolution of the QD structure and removal of the ZnS shell coating, allowing for the release of free Cd. Different surface charges due to the addition of different functional groups attached to the surface of the QD may influence both the toxic pulmonary response and the persistence and clearance of the Cd from the lungs. QD-NH2 appeared to induce more injury and inflammation at earlier time points post-exposure where epithelial cell necrosis and type II cell hyperplasia were minimal but present; whereas QD-COOH cause greater injury and cellular influx at the later time points post-exposure. Although differences exist between the two particles, the pattern of toxicity was primarily the same for both QD with toxicity peaking at days 7 and 14 and beginning to subside over time. This is in agreement with Clift et al. , who found that QD with organic coatings were more toxic than QD-COOH and QD-NH2(PEG) with only slightly more toxicity present with the carboxyl groups versus the amine groups. In addition, toxicity appears to be dependent on the availability of free Cd for both QD, as well, although dissolution rates may vary. Overall, despite a relatively high pulmonary load, the systemic availability of the QD appears to be low. No free Cd was measured in blood, brain, liver, and heart in the current study. Measureable, but relatively small, amounts of free Cd did accumulate in the kidney, the main storage organ of Cd in the body.
Overall, both forms of QD induced lung damaged which was more severe one week after exposure, with QD-NH2 having induced more toxicity at the early time points post-exposure and QD-COOH having been more toxic at the later time points. However, the peak damage for both QD is around day 7 and sustained until day 14. This damage coincided with the lack of fluorescence in lung tissue under confocal microscopy, which indicates destabilization of the QD, and with evidence that Cd still persisted in the lungs (silver staining of tissues) at the times of peak lung damage. The data suggests that Cd became bioavailable over time due to destabilization of the QD in the lung, and induced injury as it became available and while it persisted there. Althought the rate of clearance of the QD-NH2 and QD-COOH differed, indicating a potentially different destabilization rate, the pattern of clearance over time was similar, primarily via the lymphatics and translocation systemically to the kidneys.