This study is an extension of our previous study, which was aimed at understanding the chemical fate of QDs in vivo. While there has been no lack of studies regarding the fate and biological safety of QDs, many of these studies were conducted in vitro
[16–21]. Because cultured cells do not metabolize complex chemicals, in vitro cultures can not replicate complicated in vivo systems. Therefore, studies examining the actual chemical fate of QDs in intact biological systems (animal studies) are needed. This information is critical for the evaluation of the toxicity of QDs and other nanoparticles.
QDs exhibit narrow emission profiles and can emit clear and bright colors based on their strong fluorescence intensities. A fluorescence spectrometer was used to reveal some of the parameters (size and concentration) of QDs. When QDs degrade (loss of surface atoms and decreases in the size of QDs), blue-shifts in the excitation fluorescence spectra and decreases in the quantum yield are observed . However, using fluorescence intensity to quantify QDs in blood and tissues has been deemed to be problematic due to the high and variable background fluorescence that result from native blood and tissues. Moreover, the fluorescence of QDs is susceptible to environmental factors. For example, the PLQY has been demonstrated to change drastically as a result of surface chemistry, rearrangement of surface ligands, photoenhanced oxidization, and solvent effects. Any of these parameters can potentially cause large deviations in fluorescence measurements . Therefore, using fluorescence intensity and peak wavelength in order to determine the chemical fate of QDs in vivo was deemed to be problematic. Inductively coupled plasma-atomic emission spectrometer (ICP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS) are highly sensitive methods that can be used to detect most elements. For example, the detection limit for Cd can approach 0.1 ng/ml using ICP-MS. Fischer et al.  first tracked QDs via Cd measurements in the blood and organs of rats using ICP-AES and presented a quantitative report on the kinetic parameters and biodistribution of QDs in the rat. Several subsequent biological fate studies of QDs in vivo using ICP-AES and ICP-MS have been reported [12–14]. However, the ICP-AES or ICP-MS analytical techniques were unable to distinguish whether Cd was bound within the QDs or had been released into the tissues. This distinction is important because bound Cd has no valence charge and, thus, is most likely much less biologically reactive or toxic than highly charged free Cd2+.
In the work conducted by Fischer’s group , QDs were incubated with whole blood, and samples were retrieved and centrifuged at various times in order to investigate whether the QDs interacted with blood components. The supernatant (plasma) was analyzed for QDs using fluorescence spectroscopy and ICP-AES, whereas the pellet was further analyzed using fluorescence microscopy. The authors verified using optical microscopy that the QDs exhibited minimal, nonspecific binding to the cellular blood components of the rat (e.g., erythrocytes). Other studies  mimicked the pHs of blood (pH 7.4) and the renal tubules (pH 4.8) in order to investigate the stability of CdSeS/SiO2 QDs that had been stored in a buffer for five days. The results revealed that the maximal emission of CdSeS/SiO2 QDs was not altered and that the fluorescence intensity was stable after five days of incubation in either pH 7.4 or pH 4.8 buffer solutions. Because the concentration of Cd in the supernatant of the experimental solution in both pH environments did not significantly differ from that in the phosphate buffered saline (PBS) blank, the authors concluded that Cd concentrations could be used to estimate the concentration of QDs in their in vivo studies. However, in vivo toxicity (or perceived toxicity) of the high surface area-to-volume ratio of QDs, which provides a large available surface for enzymatic degradation and the release of metallic ions, has been previously reported. Fitzpatrick et al.  studied the persistence of CdSe/ZnS QDs that had been coated with mPEG 5000 (emitting at 655 nm) in mice. They noted that immediately after injection into the tail vein, emissions related to the QDs were observed in the circulatory system and various organs, including the liver, spleen, lymph and bone marrow. The fluorescence signals observed were found to have been blue-shifted from the initial emission wavelengths, due to the loss of one monolayer of the nanoparticle. Within five days, the emission observed in the liver had faded, indicating that extreme degradation of the QDs had occurred with concomitant release of Cd. Pi et al.  labeled mouse embryonic stem cells (ESCs) with QDs and monitored QD-labeling using fluorescence microscopy for 72 h. A rapid loss of QD-labeling in ESCs was observed within 48 h. Transmission electron microscopy (TEM) analysis showed a dramatic decrease in QDs in the vesicles of ESCs at 24 h post-labeling, suggesting that the QDs may have degraded.
Here, we used fluorescence spectrometer and ICP-MS to investigate the chemical fate of CdTe/ZnS aqQDs that had been stored in a buffer (pH 7.4) for 3 days (Table 1). The experimental data indicated that the maximal emission was not altered. The concentrations of Cd, Te and Zn ions released from the CdTe/ZnS QDs gradually increased over time, but were extremely low, and the ratios of Cd:Te and Zn:Cd did not vary significantly. This suggests that the size and composition of CdTe/ZnS aqQDs stored in buffer did not differ significantly from those kept in situ
[22, 23]. On the other hand, the fluorescent intensity was reduced after a 6 h incubation in a pH 7.4 buffer solution. This suggests that the nanocrystals of CdTe/ZnS aqQDs formed surface defects in a pH 7.4 environment.
Next, the Cd and Te concentrations in the blood were measured using ICP-MS, the Cd:Te ratios were calculated, and Cd and Te blood kinetic analyses were conducted. As described in Tables 2 and 3, we observed differences in the Cd:Te ratios and Cd and Te blood kinetic parameters, suggesting that CdTe/ZnS aqQDs break down in the blood. If CdTe/ZnS aqQDs are chemically stable in the blood, steady or unchanged Cd:Te ratios in the blood over time would result. Furthermore, the blood kinetic parameters, CL and t
of Cd and Te would be similar. As shown in Tables 1 and 2, the relative fluorescent intensity of QDs in pH 7.4 buffer solution was reduced rapidly after 6 h. The Cd:Te ratios also varied significantly after 6 h in the blood. These findings indicate that the stability of CdTe/ZnS aqQDs in the blood of the mice is comparable to the stability in buffer solutions (pH 7.4) . QDs exhibited minimal, nonspecific binding to the cellular components of mice blood .
Lastly, we examined the uptake of CdTe/ZnS aqQDs in various organs after leaving the bloodstream using ICP-MS. Contrary to their relatively transient fate in the blood, Cd and Te are preferentially distributed into organs and tissues (Figure 3A and B). When the specific tissue concentrations of Cd and Te were examined, Cd was observed to mainly accumulate in the liver and kidneys, which was in agreement with previous reports [13–15, 25]. Unlike Cd, the kidneys appeared to be the major target organ for Te deposits. No significant changes in Te accumulation were observed in the heart, liver, spleen and lungs. On the other hand, we found that the Cd:Te ratios in various organs over time differed. Specially, at 1 h post-injection, significant changes in Cd:Te ratios were observed in the liver and kidneys. These findings further indicated that CdTe/ZnS aqQDs degraded in specific organs, especially in the liver and kidneys , and implied that the chemical stability of CdTe/ZnS aqQDs in vitro can not mimic the biological responses or consequences that occur in vivo. As shown in Table 1, CdTe/ZnS aqQDs are stable (the maximal emission and relative fluorescence intensity of CdTe/ZnS aqQDs were not altered) after a 1 h incubation in pH 7.4 buffer solutions.
Metallothionein (MT) is a protein that is inducible by various metallic elements, especially in the liver and kidneys. Cd is a potent inducer of MT, and Cd-MT is stable and stored. MT-bound Cd complexes are believed to be responsible for the long biological half-life of Cd in the body [27, 28]. It was previously demonstrated that only free Cd release from QDs can induce MT . In our study, over a period of 28 d, we found that 42.94 and 42.60 ng/g Cd remained in the liver and kidneys, suggesting that either significant disintegration of CdTe/ZnS aqQDs had occurred, or that Cd had been released in the liver and kidneys and had transferred Cd as a Cd-MT complex during this time period. In the kidneys, a trend of increasing tissue concentrations was observed at 28 d (Figure 3A). The reabsorption of Cd indicates that renal elimination of Cd is more difficult . The half-life of Cd in the kidneys appears to be very long. In the present study, very little Cd was distributed in the brain. The brain is a challenging organ for drug delivery because the blood brain barrier (BBB) functions as a gatekeeper guarding the body from exogenous substances. Small QDs may be transferred through a space of 20 nm that separates the capillary endothelium from the astrocytes, or QDs may interact with the receptors located at the BBB .
How the stable covalent bonds of QDs were broken biologically in the tissue remains unknown and requires further investigation. If the dissociation of Cd from QDs is closely associated with the toxicity of QDs in the tissues, knowing how to actually define the stability of QDs in vivo may inspire the development of new measures to inhibit or lessen such degradation, allowing QDs to remain in use medically, but in a much safer manner.