SEMICONDUCTOR QUANTUM DOT TOXICITY IN A MOUSE IN VIVO MODEL
Svetlana V. Bozrova1, Maria A. Baryshnikova1,2, Igor Nabiev1,3, Alyona Sukhanova1,3
1 Laboratory of Nano-Bioengineering, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 115522 Moscow, Russia
2 Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, 115478 Moscow, Russia
3 Laboratoire de Recherche en Nanosciences, EA4682-LRN, Université de Reims Champagne-Ardenne, 51100 Reims, France
Quantum dots (QDs) are highly fluorescent inorganic semiconductor crystals with a diameter from 2 to10 nm (1). They can consist of a semiconductor core (CdSe, CdS, CdTe, InP, InAs, or PbSe) alone, or have a core/shell structure (usually, with a ZnS shell), the shell protecting the QD from oxygenation and enhancing the photoluminescence quantum yield (2). Regarding optical properties, semiconductor QDs are characterized by an exceptionally bright photoluminescence and rock-solid photo- and chemostability. They have broad quasi-continuous absorption and narrow, sharp emission spectra with an approximately Gaussian shape and large (>100 nm) Stocks shifts (3). The high brightness of fluorescent QDs is a result of high molar adsorption coefficients (several times higher than those of fluorescent dyes and proteins) combined with a high quantum yield (4). Moreover, different populations of QDs can be excited at the same wavelength, which can be very far from their respective emission bands, depending on the QD core size and composition. The size of QDs can be varied in the process of their synthesis, and the QD fluorescence color depends directly on their diameters.
In recent years, QDs have been increasingly widely used in biology for cell targeting, imaging, and drug delivery due to their unique optical and physicochemical properties (5). They are likely to replace the commonly used organic dyes because of their considerable advantages over the organic fluorophores (6).
In addition, QDs are increasingly used in clinical medicine. Their most promising potential applications are in cancer diagnosis, including in vivo tumor imaging.
Cancer remains one of the leading causes of death in the world (7). One of unsolved problems is differentiation of tumor-affected tissue from healthy tissue during surgery. In addition, noninvasive determination of metastases is highly demanded. These problems can be solved by using in vivo tumor imaging. Many researchers address the issue of selecting the best method for in vivo tumor imaging using different substances, such as fluorescent proteins (8–13). Advanced optical characteristics compared to organic dyes determine the necessity of investigation of different aspects of QD use for in vivo optical imaging of tumors. In this connection, the behavior of QDs in living organisms is of special interest. Certainly, the main questions are whether or not QDs are toxic for humans and, if yes, what concentration of QDs is relatively harmless.
We have carried out in vivo experiments with fluorescent semiconductor CdSe/ZnS core/shell QDs, which are currently the most widely used in research. QDs coated with trioctylphosphine oxide (TOPO) were synthesized, solubilized, and modified with electrically neutral derivatives of polyethylene glycol (PEG) containing both thiol and carboxyl groups to make them soluble in water and aqueous buffer solutions and protect them from clustering (14).
Several stages of purification after coating QDs with PEG were performed so as QDs not to bear any toxic admixtures, such as unbound PEG or other substances. The stability of QDs was accurately measured during one month before the experiments in three different solutions, phosphate buffer, RPMI medium, and mouse blood serum, at different temperatures: room temperature and animal body temperature (37°C). Since QDs were stable under all these conditions, we could be sure that they would not aggregate or decompose shortly after they enter a living body. In addition, the physical characteristics of QDs, including their sizes and emission and absorption spectra (Figures 1, 2), were measured before in vivo experiment.
Figure 1. The absorption spectrum of CdSe/ZnS core/shell quantum dots.
Figure 2. The emission spectrum of CdSe/ZnS core/shell quantum dots.
70 DMA/B6 hybrid mice were used to estimate the QD toxicity in vivo. A QD solution was injected intravenously. Pure solvent and PEG solution were used as negative controls. The mice were examined for about one month after QD injection. The survival rate was estimated throughout the study. Only in the group with the highest concentration of QDs did animals start to die during this period (survival rate, 75%).
During the experiment, we visually accessed the state of health of every animal and measured its weight (Figure 3). No significant weight loss was observed during four weeks.
Figure 3. Animal weight assessment during the experiment.
Special attention was paid to the state of the animals’ hair and eyes. It was found that mice tolerated QDs well even at a concentration as high as 10 mg/kg. The mouse hair, teeth, and eyes looked like those of healthy animals (Figure 4).
Figure 4. A mouse before the experiment (left) and a mouse three weeks after the injection of 10 mg/kg of QDs (right).
One month after the injection, the internal organs were investigated. We used paraffin sectioning and haematoxylin–eosin staining to assess the state of the liver and kidney every week during four weeks. Their morphology was the same as in healthy control mice (Figure 5).
Figure 5. Liver and kidney haematoxylin–eosin staining at the fourth week of the experiment.
These results allow us to conclude that CdSe/ZnS QDs have no considerable toxic effect in the mouse in vivo model at concentrations up to 10 mg/kg, because there were no changes in organ morphology, physical parameters, and visually assessed general condition of mice and no weight variation during the four weeks of the experiment.
However, the QDs were found to be toxic at the highest concentration studied, 20 mg/kg body weight, whith two-thirds of the animals dying shortly after the experiment was started. Further investigations will be performed to compare the single doses used in this experiment and the minimum therapeutic dose for these mice, so that we be able to use this mouse model for testing clinical applications of QDs.
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