On the forefront of research in nanobiotechnology is the exploration of potential applications for quantum dots in basic and biomedical research. Quantum dots (QDs) are fluorescent semiconductor nanocrystals that originated in the field of materials science, but their unique properties make them desirable tools for many disciplines [1
]. In the life sciences, it is hoped that their applications will permit new ways of observing, quantifying, and manipulating cellular processes that occur at the nanoscale. Much of current research is focused on using QDs to understand intracellular communication and signaling, and to create high resolution cellular images that can be used for long term in vivo
studies as well as diagnostics [2
]. QDs are particularly attractive for cancer research since they are inclined to accumulate preferentially in cancer tissue [3
]. Tumors are highly vascularized and tend to have high retention due to their lack of lymphatic drainage, allowing them to be an ideal destination for nanoparticles [3
]. In addition to a tool for diagnosis and detection, fluorescent QDs have been suggested as a possible treatment for cancer when conjugated to an antibody (cetuximab- C225) for human epidermal growth factor receptor (EGFR-1) and combined with radiofrequency field exposure. This treatment has been shown to significantly reduce cancer cell viability [5
]. For these reasons it is critical to evaluate the suitability of QDs as molecular probes and their potential effects on cells in culture, including how and where nanomaterials are internalized and ultimately, how they can be safely excreted.
QDs, compared to traditional organic fluorophores, do not photobleach as quickly over extended observation times, exhibit higher quantum yields, and offer tunable emission spectra that prevent overlap between other markers and cellular autofluorescence [2
]. QDs also tend to be brighter than traditional dyes by an order of magnitude and can be excited by any wavelength ranging from UV to red [7
]. In biological systems, QDs offer the advantage of probe stability because they are not readily degraded or metabolized in physiological environments [7
]. Although this can be beneficial for signal preservation, the heavy metal cores of QDs raise toxicity concerns. For example, cadmium exposure has been associated with kidney damage and cancer [11
]. It is currently believed that these disorders are caused by cadmium’s ability to replace zinc in biochemical reactions [11
Since the most common type of commercially available QDs are made with cadmium cores, replacements for CdSe-based QDs that can provide equivalent or superior optical properties are highly desirable and sought after [13
]. In addition to variation in core composition, QDs can be chemically engineered with a wide variety of surface modifications, matched to the particular application. When QDs designed for biological systems are fabricated with a water-soluble, protective, biocompatible coat (usually PEG or gelatin) but without reactive side chains, they are considered non-targeted [6
]. In contrast, targeted QDs are those used as probes for specific biomolecules in cells when ligands are attached to their outer coat as reactive side chains [7
We are interested in evaluating the potential of heterostructured InP QDs to serve as an alternative to CdSe-based QDs for biological applications. Although InP QDs have not yet been tested extensively for biocompatibility, the related InGaP QDs have been shown to be ~10-fold less toxic than CdSe QDs in porcine kidney cells [18
]. We designed this pilot study to determine the consequences of exposure to moderately thick-shell InP/ZnS core/shell QDs for cultured Xenopus
kidney cells (A6; ATCC). Amphibian kidney cells were selected for analysis because of the aforementioned effects of cadmium on the kidney, and also because amphibians are frequently used to assess environmental quality, including toxic effects of pollutants. The non-targeted InP QDs used in our experiments were engineered to retain brightness and emission in an aqueous environment. We compared the effects of these non-targeted InP QDs with those of commercial non-targeted QDs that previous work has shown do not enter A6 cells at exposure concentrations up to 100 nM [6
]. We reasoned that if non-targeted InP QDs have similar properties, they are good candidates for the design of targeted biomolecular probes through surface modification.
One of the most appealing aspects of using QDs as molecular probes is their capacity for visualization in both light and electron microscopes [15
]. Accordingly, we used epifluorescence and transmission electron microscopy (TEM) to evaluate morphological and ultrastructural properties of A6 cells exposed either to novel heterostructured InP QDs (LANL/CINT) or to commercially available CdSe QDs (Qtracker® 565 targeted; Qtracker® 565 non-targeted QDs; Invitrogen). Epifluorescence imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa Fluor® phalloidin; Hoechst 33342), and identification of any cells that appear associated with QDs (either internally or on the cell plasma membrane). TEM offered the unique advantage of visualizing the electron dense QDs at a higher resolution and provided the optics necessary to analyze the subcellular sequestration and compartmentalization of QDs [13
]. Our preliminary results show that cells exposed to InP QDs at concentrations up to 200 nM show comparable morphology to that of cells grown in the absence of QDs. Moreover non-targeted InP QDs can be detected in the cytosol of very few cells when delivered passively in the bath at a 200 nM concentration Taken together, our findings support the notion that these InP QDs show promise as probes for cell surface and intracellular bimolecular targets.