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. 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.
Several in vitro
studies have shown QDs to be incorporated via endocytic mechanisms by a variety of cell types. Mammalian (HeLa) and Dictyostelium discoideum
(AXS) cells were observed to incorporate avidin and DHLA-conjugated CdSe/ZnS QDs via endocytosis (Jaiswal et al. 2003
), and rat primary hepatocytes were observed to incorporate CdSe–MAA QDs (Derfus 2004
). Hoshino et al. (2004a)
observed adherence of CdSe/ZnS–SSA QDs to the surface of EL-4 cells, with subsequent endocytosis and increase in cytosolic QD concentration in a time-dependent manner (minutes to hours). Other studies have shown similar nonspecific uptake. Hanaki et al. (2003)
, exposing Vero cells to CdSe/ZnS–MUA QDs coated with SSA, observed endosomal/lysosomal localization of the QDs near the perinuclear region 5 days after exposure. Parak et al. (2002)
observed endocytosis and vesicular storage and transport of CdSe/ZnS silicon dioxide–coated QDs to the perinuclear region in human mammary tumor cells, and an in vivo
study by Dubertret et al. (2002)
demonstrated endocytosis and active transport of QD micelles (phospholipid block-copolymer) in Xenopus
In one instance, QD size was shown to be a determining factor in subcellular distribution. Lovric et al. (2005)
observed 5.2 nm cationic CdTe QDs to localize throughout the cytoplasm of N9 cells (murine microglial cell line) but not in the nucleus. In contrast, 2.2-nm cationic CdTe QDs were observed to localize in the nuclear compartment within the same time frame. Hence, in this instance, size, not charge, was a determining factor in subcellular localization. It was noted, however, that because relatively unrestrained passage of macromolecules up to 9 nm in diameter occurs through nuclear pores, the size of the QDs (2.2 and 5.2 nm) cannot be the only explanation for the entry of smaller QDs (2.2 nm) into the nucleus. Altering the bioactivity of the smaller 2.2 nm CdTe QDs by conjugation to BSA was seen to limit its localization to the cytosol.
Where nonspecific endocytic mechanisms have been shown to be instrumental in QD uptake by cells, receptor-mediated processes may also contribute to cellular internalization when QDs carry bioactive moieties specific for cell receptor types or surface proteins. Epidermal growth factor (EGF)–conjugated CdSe/ZnS QDs proved to be highly specific for the EGF receptor (erbB1), demonstrating rapid internalization into endosomes of Chinese hamster ovary cells. The endocytic vesicles were observed to undergo a directed linear motion mediated by microtubule-associated motor proteins and vesicular fusion (Lidke et al. 2004
). QDs coated with anti-Pgp showed good specificity for live HeLa cells transfected with Pgp-EGFP (EGF protein), with no apparent nonspecific cell labeling (Jaiswal et al. 2003
). Other studies yielded similar results: CdSe/ZnS QDs conjugated to peptides specific for lung, vascular, and lymphatic tissues exhibited specificity for labeling cell membranes of their targeted tissue types (Akerman et al. 2002
). Dahan et al. (2003)
found that QDs conjugated with glycine receptor (GlyR1) ligands exhibited specificity for endogenous GlyR1 subunits on cultured spinal neurons. Last, prostate-specific membrane antigen–conjugated QDs specifically targeted prostate tumors in mice (Gao et al. 2004
), and QDs complexed with a viral (SV40) nuclear localization signal peptide were observed to readily enter the nuclear compartment of human HeLa cells (Chen and Gerion 2004
In invertebrate cell types, Kloepfer et al. (2003)
observed transferrin-conjugated CdSe QDs to enter S. aureus
bacterial cells, which do not endocytose but rely on membrane transporters. The transferrin-conjugated QDs also showed clear internal labeling in the fungi Schizosacharomyces pombe
and Penicillium chrysogenum
. No internal labeling of nonpathogenic staphylococci and micrococci was observed, and it was suggested that transferrin-mediated transport processes were involved in cell-specific uptake.
Importantly, where endocytic mechanisms have been observed in a variety of cell types, the question of systemic distribution arises. Although few in vivo
studies exist, they suggest that QDs may be systemically distributed in rodent animal models and accumulate in a variety of organs and tissues. EL-4 cells containing CdSe/ZnS–SSA QDs (via endocytosis) were observed in the kidneys, liver, lung, and spleen of mice up to 7 days after injection, with spleen and lung having the most accumulation (fluorescence) (Hoshino et al. 2004a
). Similarly, Ballou et al. (2004)
, employing QD coatings of different molecular weights [MW; methoxy-terminated PEG, MW 750 (mPEG-750), carboxy-terminated PEG, MW 3,400 (COOH–PEG-3400), and ethoxy-terminated PEG, MW 5,000 (mPEG-5000)], observed differential tissue and organ deposition in mice in a time- and size (MW)-dependent manner. For instance, mPEG-750 QDs and COOH–PEG-3400 QDs were cleared from circulation by 1 hr after injection, whereas mPEG-5000 QDs remained in circulation for at least 3 hr. At 24 hr after injection, mPEG-750 QDs were observed in the lymph nodes, liver, and bone marrow. In contrast, significantly less retention of COOH–PEG-3400 and mPEG-5000 QDs was observed in lymphatic tissue compared with bone marrow, liver, and spleen. At 133 days, continued fluorescence of mPEG-750 QDs was observed in the lymph nodes and bone marrow. A study by Akerman et al. (2002)
yielded comparable findings. Lung- and tumor-targeting peptide-coated CdSe/ZnS QDs injected into mice (iv), regardless of the peptide used for the coating, accumulated in both the liver and spleen in addition to the targeted respiratory tissues. Interestingly, additionally coating CdSe/ZnS QDs with PEG (a polymer known to minimize molecular interactions and improve colloidal solubilities) nearly eliminated the nonspecific uptake of QDs into the liver and spleen. An in vivo
study employing Xenopus
embryos revealed that QDs, once internalized by cells, subsequently may be transferred to daughter cells on cell division. Dubertret et al. (2002)
, injecting a CdSe/ZnS micelle (PEG–PE and phosphatidylcholine) conjugated with an oligonucleotide into Xenopus
embryos, observed QD labeling of all embryonic cell types, including somites, neurons, axonal tracks, ectoderm, neural crest, and endoderm. The internalized QDs were localized to both the cytosol and nuclear envelope and were transferred to daughter cells on cell division. The progeny of the QD-injected cells were shown to contain fluorescent QDs after several days of development.
Metabolic processes and excretory mechanisms involved in the elimination of QDs, as well as in vivo
bioactivity, remain poorly understood and have not been well studied. 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: the liver, spleen, and lymphatic systems. However, this a rough generalization, and discrepancies in the literature exist. For instance, subcutaneous injection of CdSe/ZnS–PEG-coated QDs in mice showed clearance of the QDs from the site of injection, with accumulation of QDs in lymph nodes. In contrast, Akerman et al. (2002)
observed that modification of lung-and tumor-targeting peptide-conjugated CdSe/ZnS QDs with a PEG coating nearly eliminated nonspecific elimination of QDs via the lymphatic system.
The above studies suggest that QDs may see variable systemic distribution dependent on individual QD physicochemical properties. Although studies are limited, QD tissue/organ distribution seems to be multifactorial, depending on QD size, QD core–shell components, and the bioactivity of conjugated or otherwise attached functional groups. Size alone can markedly affect distribution kinetics, and QD surface coating can govern serum lifetime and pattern of deposition (Ballou et al. 2004
; Lovric et al. 2005
). QDs lacking specialized functional groups or specificity have been shown to be incorporated via endocytic mechanisms by a variety of cell types, both in vivo
and in vitro
. In contrast, QDs bearing natural ligands specific for cell receptors and cell membrane proteins have been shown to be specific for given cell membrane proteins/receptor types. Several studies have shown nonspecific QDs to adhere to cell surfaces, possibly through interactions of QD with glycoproteins and glycoplipids in cell membranes. Although many studies indicate endocytic processes and intracellular vesicular trafficking and storage of QDs, the exact mechanisms remain to be elucidated. Subcellular localization is variable, like systemic distribution, and dependent on QD physicochemical properties. Such variables, determined by the unique physicochemical properties of individual QD types, will prove significant in developing characterization protocols for QD toxicity screening, given the nonuniformity in size, QD functional coatings, core–shell complexes, and outer coating photolytic and oxidative stability.