Abstract: Quantum Dots (QDs) are autoflorescence semiconductor nanocrystals that can be used for in vivo biomedical imaging. However, we know a little about their in vivo distribution in tissue organs and health consequences. The aim of this study was to detect QDs biodistribution in different organs from healthy female and male mice after single intravenous injection at the dose of 2.98 pmol CdSe/CDs/ZnS QDs/mouse for up to 14 day in female and 8 h in male mice. Laser scanning confocal microscope and/or florescence light microscopy was used to detect QDs in different samples. The results revealed that most of QDs were highly accumulated in spleen, liver, lung of treated mice; however, small amount of QDs was detected in kidney. There is no QDs were observed in other organs such as heart of female mice and brain of male mice of treated group. We also didn’t find QDs in all samples prepared from control group and blood sample of treated mice at different time points. Effective and rapid (1 h) detection of tissue organs and blood samples using fluorescent imaging of quantum dots was demonstrated. This work was done using a very low dose (2.98 pmol/mouse) of injected QDs.
INTRODUCTION
Quantum Dots (QDs) are fluorescent inorganic nanometer sized crystals with remarkable unique optical and electronic properties. They are composed of a fluorescent core of semiconductor heavy metals. In order to limit their potentially toxic effects due to the release of Cd2+ or Se2- ions in case of oxidation of the core by ultraviolet light and to passivate the defects on its surface, this core is encapsulated in a shell of organic material, polymeric or lipid-based layers then coat the shell. The QDs are emerging as a new class of luminescence probes for biology and medicine research applications. The QDs possess unique optical tunability, such as size-tunable emission wavelength, superior signal brightness, resistance to photobleaching and broad absorption spectra for simultaneous excitation of multiple fluorescence colors using a single excitation source (Michalet et al., 2005; Medintz et al., 2005; Robe et al., 2008).
Several studies have shown QDs may be systemically distributed and may accumulate in organs and tissues. Absorption, Distribution, Metabolism and Excretion (ADME) characteristics are, not surprisingly, highly variable for QDs because of the wide variation in QD physicochemical properties. The QD size, charge, concentration, stability and outer coating bioactivity each contribute to not only the potential toxicity of a given QD but also to their ADME characteristics. Physicochemical properties in conjunction with environmental factors and QD stability (oxidative and photolytic lability) together are a paradigm in which ADME characteristics of QDs can be highly variable and difficult to predict (Hardman, 2006). QDs have been already applied to visualize SLN in gastrointestinal tract (Soltesz et al., 2006), pleural space (Parungo et al., 2005a), lung area (Soltesz et al., 2005), oesophageal area (Parungo et al., 2005b), skin (Tanaka et al., 2006) and axilla (Kim et al., 2004) using infrared light. Recently, red and infra-red emitting QDs were imaged in inguinal and axillary lymph nodes in tumor bearing mice already 3 min after local injection (Ballou et al., 2007).
Currently, only few detailed works on QDs biodistribution in rodents have been realized (Fischer et al., 2006; Gopee et al., 2007; Choi et al., 2007; Robe et al., 2008). The QDs toxicity study is ultimately related to the investigation of their biodistribution in pre-clinical models. More importantly, one can visualize the labeling dynamic process of the bioconjugated QDs in the cellular level using appropriate imaging setup such confocal microscopy and multi-photon imaging techniques (Yong et al., 2007, 2009).
The main objective of this study is to observe the QDs biodistribution in the different organs of both male and female post-injection via tail veins using fluorescence and confocal microscopy.
MATERIALS AND METHODS
Experiments of this study have been carried out during 2008 and 2009.
Synthesis of QDs
The CdSe/CDs/ZnS QDs were prepared by growing a CDs/ZnS-graded shell on
a CdSe core. Briefly, the reaction was carried out with CdSe core QDs corresponding
to 0.3 g, using a Cd: Zn: S ratio of 1: 3: 4 (0.5 mmol Cd, 1.5 mmol Zn and 2
mmol S) and 10 mL of oleic acid at 300°C. The CDs initially prefers to grow
on the CdSe dots because the CdSe lattice mismatch with CDs is less than that
of ZnS. The CDs layer will mediate the growth of the more strained ZnS. The
shell growth is uniform and epitaxial and eventually coats the CdSe core. The
QDs were separated from the surfactant solution by the addition of ethanol and
centrifugation. The reddish QD precipitate could be readily redispersed in various
organic solvents (hexane, toluene and chloroform).
Preparation of Lysine Cross-Link Mercaptoundecanoic Acid Coated QDs
Typically, 3 mmol mercaptoundecanoic acid (MUA) was dissolved in 10 mL of
chloroform under vigorous stirring. After stirring for 10 to 15 min, 2 mL of
concentrated (~30 mg mL-1) CdSe/CDs/ZnS QD solution was added into
this mixture. Approximately 1 min later, 2 mL of ammonium hydroxide was added
to the vigorously stirring solution then was stirred overnight at room temperature.
The QDs were separated from the surfactant solution by addition of ethanol and
centrifugation. The QD precipitate was re-dissolved in 15 mL DMSO for further
lysine cross-linking process. The lysine cross-linked MUA QDs were obtained
by mixing DMSO quantum dots solution with both DCC (~30 mmol) and lysine (~15
mmol) under vigorously stirring for 2 h. The lysine coated QDs were precipitated
from the solution by addition of ethanol and centrifugation. The red-brownish
precipitate was redispersed in 10 mL HPLC water and the solution was further
filtered using a syringe filter with a nominal pore diameter of 0.45 μm.
The lysine coated QDs have relatively good colloidal stability and no precipitation
was observed after several months. This stock solution was kept in the refrigerator
at 4°C for further use.
Animals
Four-week-old male and female prepubertal kunming mice were purchased from
Guangdong Medical Lab. Animal Centre, China. The mice were acclimated for 2
weeks in the animal facilities at the life science school (Shenzhen, University).
Mice were kept in 12 h light/dark cycle and had access to food and water ad
libitum. Animal procedures were performed in compliance with institutional
and national guidelines.
Experimental Groups and QDs Administration
In the present study, two types of experiments were carried out: first experiment,
a total of 30 male mice and the second experiment, 42 female mice (six weeks
old), weighing from 28 to 31 g were used in this study. Two groups of animals,
each composed of 15 mice, were used for biodistribution studies in male mice:
each mouse of control group injected with 100 μL of saline buffer and treated
group injected with 100 μL containing 2.98 pmol QDs/mouse via tail veins.
Three time points were use to check the QDs biodistribution in different organs
2, 4 and 8 h for male mice and 1, 2 and 14 day for female mice after the injection.
At the end of each time point, 10 male mice (5 each group) and 14 female mice
(7 each group) were killed. After sacrifice, blood samples were separately collected
from each mouse. All organs of each mouse including spleen, kidney, heart, lung,
brain, liver and testis for males and ovaries for female were removed and made
soluble for slides preparation.
Microscopic Investigations
All images were obtained by laser scanning confocal microscope (TCS SP2,
Leica, Germany) and/or florescence light microscopy. The excitation wavelength
used in this study is 488 nm. Both reflection and fluorescence images were obtained
for all organs and acquired simultaneously using the two-wavelength band-selected
channels. The wavelength band setups for collecting reflection and fluorescence
images were 480-500 and 580-620 nm, respectively. Fluorescence Microscope (Olympus
IX71, Japan) was used to obtain images too.
RESULTS
QDs Biodistribution in Female Organs
Female mice were injected with QDs, scarified at 1, 7 and 14 day after injection,
all organs removed and slides were prepared from different tissue organs. Results
showed that QDs have been detected in spleen, liver and lung at different time
points.
| Table 1: | QDs biodistribution in different organs of female mice (day as a time point) |
| -: No QD has been detected, +: Small amount, ++: Medium amount, +++: High amounts of Qds | |
| Fig. 1: | Examples for confocal and fluorescence microscopy images from tissue organs of female mice 1, 7 and 14 d after single QDs injection via tail veins |
However, QDs have been detected in brain, ovaries and kidney at 7 day after injection. On the other hand, there is no QD has been found in all samples of control group and blood samples from all treated group at different time points (Table 1, Fig. 1).
Qds Biodistribution in Male Organs
In this experiment, as shown in Table 2 and Fig.
2 no QDs have been detected in all samples from control group, brain and
blood samples of treated group at all time points. The QDs accumulated in spleen,
liver and heart. However, a small amount of QDs has been detected in both testes
and kidney from treated group at 8 h post-injection.
| Table 2: | QDs biodistribution in different organs of male mice (hour as a time point) |
| -: No QD has been detected, +: Small amount, ++: Medium amount, +++: High amount of Qds | |
| Fig. 2: | Examples for confocal and fluorescence microscopy images from tissue organs of male mice 2, 4 and 8 h after single QDs injection via tail veins |
DISCUSSION
In the present study, the injected mice were viable and healthy until the end of each experiment. Moreover, no noticeable ill effects in mice injected with QDs. In an in vivo study employing mice, Ballou et al. (2004) injected (iv) amphiphilic polyacrylic acid polymer-coated QDs (amp-QDs) and amp-QDs conjugated to PEG-amine groups (mPEG-QDs), at QD concentrations of 20 pmol QD/g animal weight. Necropsy showed no signs of necrosis at the sites of tissue deposition and injected mice were viable for 133 day until the time of necropsy. No obvious sign of QD breakdown in vivo was detected by electron microscopy (in vivo QD stability was presumed to be due to the amphiphilic polymer coating). In another in vivo study, Larson et al. (2003), observed no noticeable ill effects in mice injected (iv) with 20 nm and 1 μm solutions of CdSe/ZnS QDs (ill effects was not defined).
In general, QDs distribution depends on their physicochemical properties and in particular, size and coating. Tracer particle size has an important effect on migration time. Particles less than 5 nm diffuse into blood, those between 5 to 10 nm can migrate through nodal tissue and result in false-positive results and those higher than 1000 nm largely remain at the injection site (Parungo et al., 2005a; Tanaka et al., 2006). We used QDs coated with lysine with a size ranged from 20 to 25 nm and the QDs were detected at 2, 4 and 8 h post-injection in male organs and 1, 7 and 14 day in female organs, especially in spleen, liver and lung. It has been reported that QDs with a diameter of 16 nm, since a size range between 15 and 20 nm is optimal to enable the QDs to travel through the lymph channels and be trapped by SLN (Robe et al., 2008). The distribution in healthy mice of 48 pmol intradermally injected QDs with a diameter of 37 nm and PEG coating has been investigated by Gopee et al. (2007). The authors did not find any QDs in lymph nodes until 12 h after injection, whereas Robe et al. (2008) could detect them already 5 min following the injection. An early detection (3 min) of QDs traveling toward inguinal nodes was observed after intratumoral injection of red emitting QDs at concentrations similar to ours (Ballou et al., 2007). However, this observation was not followed by biodistribution investigations. The QDs fluorescence increases with time to reach a maximum at 60 min after the injection.
Data obtained from the present study showed that most of QDs were highly accumulated in spleen, liver, lung from male and female mice; however, small amount of QDs was detected in kidney and there is no QDs were observed in other tissue organs such as heart of female and brain of male mice. In contrast to our results, the previous QDs biodistribution studies all evidenced the presence of QDs in the liver and also to a minor extent in spleen and kidneys (Fischer et al., 2006; Gopee et al., 2007). In vivo studies suggest that, regardless of the specificity of the QD, vertebrate systems tend to recognize QDs as foreign, with elimination of the materials through the primary excretory organs/systems.
CONCLUSION
In this study, we demonstrated that this rapid and selective accumulation of QDs in different tissue organs of female and male. Effective detection of tissue organs and blood samples using fluorescent imaging of quantum dots was demonstrated. This study was done using a very low dose (2.98 pmol/mouse) of injected QDs.
ACKNOWLEDGMENTS
This study was supported by grants from the National post-doctor Foundation of China (20080440780), Shenzhen/Hongkong Innovation Circle (CXQ2008043), the company of Combest of Guangzhou, Guangdong Province Science Foundation (NO. 2008078) and SZU R/D Fund (2005027, 4wxmA, 000013).