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THE CHEMICAL RECORD

Single-Molecule Measurements With a Single Quantum Dot NORITADA KAJI,1,2 MANABU TOKESHI,1,2 YOSHINOBU BABA1–5 Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan 2 MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya 464-8603, Japan 3 Plasma Nanotechnology Research Center, Nagoya University, Nagoya 464-8603, Japan 4 National Institute of Advanced Industrial Science and Technology, Takamatsu 761-0395, Japan 5 Institute for Molecular Science, National Institutes of Natural Sciences, Myodaiji Nishigo-naka 38, Okazaki 444-8585, Japan

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Received 21 June 2007; Revised 15 September 2007; Accepted 10 August 2007

ABSTRACT: Recent progress of quantum dot (QD) applications in single-molecule measurements are reviewed in this paper. Bright fluorescence and anti-photobleaching properties of QDs have explored the way to conduct long-time trajectory tracking of transmembrane proteins both in vitro and in vivo. Coupled with diversities of chemical and biochemical modifications of QD surfaces, their application fields are expanding to multidiscipline fields including imaging on the basis of a single molecule. Currently, molecular interactions and conformational changes on the QD surface can be detected at a single-molecule level. These expansions of application fields also involve toxicity problems in cells since most commercially available QDs consist of cadmium selenide or cadmium telluride, which are inherently toxic. For widespread applications of QDs including in vivo and therapeutic use in place of current organic fluorophore, cytotoxicity is discussed as well in this paper. © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 7: 295–304; 2007: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20128 Key words: quantum dots; single-particle tracking; fluorescence resonance energy transfer; cytotoxicity; single-molecule analysis

Introduction Semiconductor nanocrystals, quantum dots (QDs), are an excellent example of nanomaterials that show their potential ability brought by nanotechnologies. Tremendous physicochemical properties of QDs such as bright photoluminescence, narrow emission, broad absorption wavelength, and high photostability are attributed to their small size, shape, and chemical composition. As an alternative to organic fluorescence dyes, there has been increasing use of QDs in medicine for purposes The Chemical Record, Vol. 7, 295–304 (2007) © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

of diagnosis, imaging, and gene delivery. In recent years, these unique properties of QDs have been initiating evolutional changes in single-molecule techniques. Single-molecule measurements have provided a wealth of information and have allowed better understanding of a wide range of physical,

䉴 Correspondence to: Noritada Kaji; e-mail: [email protected]

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chemical, and biological phenomena and processes. The technique of single-molecule analysis has the ability to resolve molecular-scale heterogeneities which seem to be homogeneous samples from a macroscopical viewpoint in conventional ensemble measurements. QDs can be observed at a singleparticle level and can be tracked over an extended period of time in live cells with conventional optical detection systems such as confocal microscopy. Single-particle tracking,1 which is one of the powerful tools for investigating membrane protein properties in live cells, will receive the benefits by introducing QDs as a new probe instead of gold nanoparticles, polystyrene beads, and organic fluorophores. In single-particle tracking, even the random intermittency of QD fluorescence emission could help to identify the tracked QD as itself and to distinguish aggregates from a single QD. In the first part of this paper, we review the current reports based on single QD tracking. High photostability and high degree of flexibility of QD surface modifications are useful for single-particle fluorescence resonance energy transfer (FRET) to detect conformational changes of QD surface molecules over a long period of time.

Single-particle FRET has enabled heterogeneities of molecules to be seen and kinetic parameters such as enzymatic reactions to be estimated. Having said that, detection of a single-particle FRET signal is relatively easy because it could be handled as on and off signals. On the contrary, since we do not know what interactions exist on the nanoscale curvature on a QD and what conformation the surface molecules show, kinetic parameters of reactions on a QD surface should be carefully estimated considering the Förster radius. The study regarding this matter is also reviewed consequently. Highly diluted QDs are applied in single-molecule analysis; nevertheless, cytotoxicity of QDs must be carefully considered. In the final section, a major concern about QDs in in vivo imaging and therapeutic applications, cytotoxicity, is discussed. According to the rapid development of nanotechnology, nanotoxicology becomes an emerging discipline in which humans experience inhalation, ingestion, skin uptake, and injection of nanoscaled materials. Gold nanoparticles, for instance, are the most widely used nanoparticles in the biological field and are believed to be inherently nontoxic. However, even the gold nanoparticles, the surface functionalization could

䉴 Dr. Noritada Kaji obtained a bachelor’s degree in Pharmaceutical Sciences in 2000 and a Ph.D. degree in 2004 from the University of Tokushima, Japan. In his Ph.D. study, he worked on nanopillar chips which are state-of-the-art 2mTAS combining nanofabricated structures for DNA analysis. After his postdoctoral research, he started working as an Assistant Professor at the Department of Applied Chemistry at Nagoya University in February 2005. Currently, he is also a researcher of MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University. His research interests are mainly divided into three parts: nanobiodevices for DNA analysis, single-molecule biophysics, and biological process on mTAS. 䊏

䉴 Manabu Tokeshi is an Associate Professor at the Department of Applied Chemistry at Nagoya University. He received his Ph.D. degree from Kyushu University in 1997. After a postdoctoral research fellowship with Japan Society of Promotion of Science at The University of Tokyo, he joined the research staff at Kanagawa Academy of Science and Technology (KAST) in 1998. He was a Sub-Leader of Integrated Chemistry Project from 1999 to 2003 and Leader of Micro Chemistry Group from 2003 to 2004 at KAST. Before joining Nagoya University in 2005, he was President at the Institute of Microchemical Technology. His research interests are in the development of micro- and nanosystems for chemical and biochemical applications. 䊏

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change their cytotoxity.2 Goodman et al. reported that cationic gold nanoparticles are moderately toxic, whereas anionic nanoparticles are quite nontoxic. They attributed this difference to concentration-dependent lysis mediated by initial electrostatic binding to the cell membrane. It is not surprising that cadmium-containing nanoparticles, QDs, may be toxic to both cell cultures and live animals. The most widely believed hypothesis about cytotoxicity of QDs is derived from the release of free Cd2+ in cells. By exposing QDs to prolonged oxidative environments, such as air or photooxidation with UV light, a structural defect is developed and subsequent Cd2+ liberation occurs. So, correct and highly stable surface coating of QDs can minimize cytotoxicity arising from optical imaging processes. Currently, multidisciplinary studies involving materials, surface functionalizations, and intracellular trafficking pathway are performed to evaluate QD cytotoxicity. In light of these new studies, it is important to distinguish whether cytotoxicity arises from the interaction of QD surface molecules against cell membrane or intracellular uptake of endocytosed QDs which accompanies degradation of QD shells.

Single-Particle Tracking Single-molecule analysis, which involves a single DNA protein,3 DNA drug,4 protein–protein interaction,5 and protein confor-

mation study,6 has become one of the strongest approaches combining with the development of nanotechnology to reveal dynamics and kinetics of biomolecule interactions at a singlemolecule level. Although observed reactions generally represent an ensemble of mass of molecular reactions, single-molecule approaches give precise clues to figure out interaction manners4 or how many steps the reaction consists of.6 Single-particle tracking (SPT) is a powerful technique to understand the dynamics of cellular organization such as membrane transport and receptor in the living cell. So far, single-molecule properties in living cells have been investigated by utilizing 40 nm gold nanoparticles, 500 nm latex spheres, or fluorescence from organic dyes7,8 as a probe to track single protein dynamics. Semiconductor nanocrystals, QDs, have several advantages over gold nanoparticles, latex spheres, and conventional fluorescent dyes. Small but bright fluorescence of QDs enables the nanoscale protein movement to be monitored without any special signal enhancements by highly sensitive charge-coupled device camera or image analysis software. High photostability of QDs also allows long-time and real-time tracking. Even their blinking property, which is generally considered as their worst weakness for SPT, could be a clue to distinguish a single QD from an aggregate. Chen et al. have used QDs conjugated with integrin antibodies and demonstrated changes in the integrin dynamics during osteogenic differentiation of human bone marrow-

䉴 Dr. Yoshinobu Baba is a professor at the Department of Applied Chemistry, Graduate School of Engineering, Nagoya University. He is also Vice Director at the Health Technology Research Center, National Institute of Advanced Industrial Science and Technology; Microdevice Group Leader, MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University; Professor at the Plasma Nanotechnology Research Center, Nagoya University, and a visiting professor at the Institute for Molecular Science, National Institutes of Natural Sciences. He serves on the boards of over 15 scientific journals, including Analytical Chemistry, Lab on a Chip, and Nanobiotechnology, as an editorial board member. He is a member of the Steering Committee for International Symposium on Micro Total Analysis System (uTAS). He is a co-initiator for the world’s largest Nanotech/Nanobio International Meeting and Exhibition in Japan. He is a general chair of several international meetings including, uTAS 2002, ISBC 2003, and Microscale Bioseparations Kobe 2005. He has been admitted as Fellow of the Royal Society of Chemistry and American Academy of Nanomedicine, and has received several awards for his contributions in capillary electrophoresis, microchip, and nanobiotechnology: Young Scientist Award from the Pharmaceutical Society of Japan in 1997, Science Award from Tokushima Newspaper in 2002, Takeda International Award for Science and Technology in 2003, Award from the Society of Toxicology in 2004, MERCK Award in 2004, and Award from the Applied Physics Society of Japan in 2006. His major area of interest is nanobioscience and nanobiotechnology for genomics, proteomics, glycomics, systems biology, single-molecule manipulation, and medical applications. He is the author or coauthor of 530 publications, including research papers, proceedings, review papers, books, and book chapters. He has delivered more than 482 plenary and invited lectures at conferences, universities, and governmental/industrial laboratories. 䊏


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derived from progenitor cells.9 Until now, the integrin dynamics especially the integrin lateral mobility has been investigated by several biophysical techniques such as fluorescence recovery after photobleaching (FRAP).10–12 However, FRAP measures the averaged integrin dynamics over a distance of micrometer

range. SPT with QDs enables the observation the integrins’ rapid lateral diffusion with a nanometer scale (Fig. 1) and the successive stages of osteogenic differentiation over 14 days. Dahan et al. studied the glycine receptor diffusion dynamics in the neuronal membrane using streptavidin-

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Fig. 1. Typical quantum dot (QD)-conjugated integrins in a cell and their trajectories by single-particle tracking. (A) A differential interference contrast image of a human bone marrow-derived progenitor cell (BMPC) was superimposed with a fluorescence image of integrins labeled with QDs (red spots) indicated by arrows. (B) Representative trajectories of integrins on the surface of human osteoblast and (C) BMPC, and (D) their mean square displacements as a function of time. The figures were reproduced with permission from Chen et al.9

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functionalized CdSe-ZnS QDs under various timescales from microseconds to minutes.13 In this study, endogenous GlyRα1 subunits at spinal cultured neuron surfaces was imaged by using mAb2b primary antibody, biotinilated anti-mouse Fab fragments, and QDs emitting at 605 nm. The diffusion coefficients were obtained by following trajectories of QD–GlyR complexes. Surprisingly, the SPT of QD-labeled GlyR could be visualized for at least 20 min, whereas those labeled with Cy3-dye-coupled antibody were visible for only 5 s. Moreover, Dahan et al. found that blinking of a single QD provided a useful signature for tracking the movement of the dot through fluorescence microscopy and that a lateral resolution with QDs of about 5–10 nm could be achieved while that with Cy3 was only about 40 nm. This is due to its high signal-to-noise ratio of about 50, almost an order of magnitude higher than the signal obtained with organic dyes. Meanwhile, Dahan et al. were concerned about cytotoxicity of continuous illumination so they acquired a time-lapse imaging of one 75 ms exposure per 1 s for 20 min. This method makes sense to suppress inherent cytotoxicity of QDs caused by long-term illumination or shell breakage as well, which will be discussed later. Direct evidence for that cystic fibrosis transmembrane conductance regulator (CFTR) showed immobilization by Cterminal PDZ (postsynaptic density protein 95/discs larte/ sonula occludens-1) interactions was obtained by the SPT technique.14 CFTR is a cAMP-regulated chloride channel expressed in plasma membranes of many epithelial cell types in the airways/lung and gastrointestinal and reproductive tracts, and its mutations are commonly caused by mutations in the CFTR gene. So far, reduced CFTR diffusional mobility was predicted if CFTR-EBP50-ezrin-actin interactions were relatively stable. By using FRAP technique, Haggie et al. demonstrated fairly rapid and unrestricted diffusion of CFTR tagged with green fluorescence protein at its N terminus after C-terminal deletion or mutation.15 However, it was still unclear which interactions did constrain the diffusion of CFTR. Haggie et al. studied CFTR mobility and interactions at a single-molecule level by time-lapse imaging and SPT of QDlabeled CFTR molecules. The measurements were performed using several mammalian cell lines expressing an externally epitope-tagged CFTR. While QD-labeled CFTR diffused in the plasma membrane within 100–200 nm, it moved over the micrometer range after several treatments that ablated the Cterminal coupling to the actin skeleton via EBP50/ezrin. Along with these in vitro studies, in vivo applications of QDs become increasingly important to analyze the pharmacokinetics of drugs and stem cell tracking in tissue engineering. Although various in vivo imaging techniques such as computed tomography, magnetic resonance imaging, positron emission tomography, and organic fluorescence or luminescence imaging have insufficient time—or spatial resolution, QD imaging enabled real-time and in vivo single-particle tracking with a high


spatial resolution of 30 nm. Tada et al. demonstrated that, even in vivo, the trajectories of single functional QDs in tumors of mice from a capillary vessel to cancer cells could be traced utilizing a dorsal skinfold chamber model and a high-resolution intravital imaging system.16 They have succeed to quantitatively analyze the rate-limiting constraints on QD-antibody delivery as the following six processes: circulation within a blood vessel, extravasation, extracellular region, binding to human epidermal growth factor receptor 2 (HER2) on the cell membrane, permeation from the cell membrane to the perinuclear region by endocytosis, and the perinuclear region. There is still very little understanding of the biological behavior of nanocarriers in vivo such as immunoliposome17 and multifunctional envelope-type nano device (MEND).18,19 Their approach is expected to bring a new insight in tumor-targeting nanocarriers to increase the therapeutic efficiency and decrease the adverse effect. In the experiments using QDs, we could not entirely exclude an influence of steric hindrance of labeled QDs on their diffusion process in SPT. With the emission wavelength varying from 400 to 1350 nm, QD size would be enlarged from 2.0 to 9.5 nm19 excluding surface modifications. When the surface modifications for solvation and labeling were considered, the diameter of the QDs ranged from 10 to 20 nm. By considering this point, Dahan et al. performed experiments with a Cy3-labelled Fab fragment of the primary antibody as a control experiment, which is a much smaller (~3 nm) and monovalent molecule. They reported comparable proportions of rapidly diffusing receptors with Cy3 and QDs.13 Having said that, particular attention should be given to the size of QDs and cross-linking ability through different molecules.

Single-Particle FRET The principle of FRET shows widespread capability for detecting hybridization20,21 or enzymatic reactions.6 Incorporated with single-molecule fluorescence detection techniques, even unamplified and low-abundance DNA molecules could be detected.22–25 On the basis of the Förster formalism, only fluorophore acceptors in close vicinity of a fluorophore donor will be illuminated by means of FRET, and as a result, specific interactions could be detected. However, especially at a singlemolecule level, accurate determinations of positive signal become increasingly difficult due to the intrinsic background fluorescence of coexistent probes. So, to minimize background fluorescence noise and to achieve a single-molecule FRET signal detection, Zhang et al. developed an inorganic/organic hybrid FRET nanosensor to detect low concentrations of DNA (~50 copies or less) in a separation-free format.26 They prepared two oligonucleotides which consist of different targetspecific sequences: a reporter probe labeled with Cy5 and a capture probe labeled with biotin for conjunction with

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streptavidin-conjugated QD. Owing to broad absorption and narrow emission wavelengths of QDs, the background fluorescence could be almost completely eliminated. Furthermore, while most of FRET-based techniques are one-to-one fluorescence transfer, the signal from sandwiched hybrids is amplified through a certain number of acceptors linked to a donor. To prevent photobleaching of fluorophore acceptors, they conducted the FRET detection in a continuous-flow manner inside a microcapillary. Using this nanosensor, Zhang et al. demonstrated the detection of a point mutation typical of some ovarian tumors in clinical samples.26,27 These unique applications raised new questions on what interactions occurred between biomolecules and biomolecule– QD conjugates. Algar and Krull explored the interactions between labeled oligonucleotides and the surface of mercaptoacetic acid (MAA)-capped QDs based on FRET efficiency.28 The following three phenomena were focused on in this research: the adsorption and conformation of oligonucleotides on MAA–QDs, the kinetics of adsorption and hybridization with MAA–QDs and QD–ssDNA conjugates, and the thermal stability of hybrids as QD–dsDNA conjugate. The adsorptive interactions were studied by modulating pH, ionic strength, addition of formamide, and differences between ssDNA and dsDNA, and finally, were rationalized in terms of a hydrogenbonding model. The kinetics of adsorption and hybridization were also dependent on the strength of adsorptive interactions. The adsorptive interaction was suggested to stabilize QD– dsDNA hybrids since the melting temperatures were elevated by 1–2˚C. These minute changes of local surroundings described herein also suggest the presence of conformational variety even under the same sequences of oligonucleotide. This resulted in the different separation distances from QD to acceptors. In fact, the long separation distances in QDstreptavidin-biotin-oligomer-acceptor conjugates constructed by Zhang et al.26 limited the energy transfer efficiency from one QD to one acceptor at about 4%. Since a high number of acceptors per QD compensated for the low energy transfer efficiency, the detected FRET signals seemed to be high enough compared to conventional organic dye-based FRET. To elucidate correlation between single particle and ensemble FRET measurements, Pons et al. observed FRET of QD-dye labeled maltose-binding protein (MBP) in terms of derived FRET efficiencies and donor–acceptor separation distances29 (Fig. 2). They performed quantitative comparison between singleparticle FRET and ensemble measurements as averaged FRET efficiencies in solution phase. The results of single-particle FRET provided information about heterogeneity in the number of proteins per QD which seemed to be a macroscopically homogeneous conjugate. Since the binding constant of QD and MBP derived from single-particle FRET was consistent with the value in ensemble measurements, single-particle FRET offers complemental information to those collected

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from ensemble measurements. For the development of specific QD-based biomolecular probes in intracellular sensing of protein interactions and protein trafficking across the cell membranes, these microscopically heterogeneous surroundings on QDs should be considered for the future design of single-particle FRET.

QD for Drug Delivery Systems (DDS) and Gene Delivery Systems (GDS) When we mentioned about particular applications of QDs in DDS and GDS focused on a single molecule, the number of report was not many so far. In order to recognize in vitro and in vivo dynamics and kinetics of drugs and genes after post injections, fluorescence-based techniques have been widely used. Especially for gene delivery, viral vectors have offered outstanding transfection efficiencies, but they have encountered problems with toxicity and immunogenicity in clinical steps. As an attractive alternative, nanocomplexes consist of plasmid DNA and cationic polymers or lipids, which tightly condense through electrostatic interactions, provide safer and efficient gene transfer.30–33 Recently, Hama et al. demonstrated that, after penetrating into the nucleus, the unpacking process of the nanocomplexes is necessary for gene expression, and they pointed out less efficiency of the nanocomplexes compared with viral vectors.34 Therefore, development of detection techniques to provide sufficient sensitivity to detect the localization and to distinguish the onset of dissociation is an urgent task. FRET, which has the ability to distinguish association and dissociation states of nanocomplexes at the nanometer scale, offered a solution to this matter. Condensation states of nanocomplexes have been described by FRET, where DNA has been doubly labeled,35,36 or polymer and pDNA have been separately labeled37 with a pair of organic fluorophores. However, these organic fluorophore-based FRETs are susceptible to photobleaching and hamper time-lapse studies of intracellular trafficking. In contrast, QDs could be observed at a single-particle level and tracked over several hours in live cells even under strong exposure by laser scanning confocal microscopy. Ho et al. demonstrated that, using QD-labeled pDNA and Cy5-conjugated chitosan, QD-FRET became one of the promising techniques in GDS study because of its highly sensitive and quantitative data throughout intracellular trafficking of nanocomplexes.38 Utilizing Hoogstein base pairing by peptide nucleic acid (PNA) and DNA, plasmid DNA was biotinylated through binding of a biotinylated peptide nucleic acid, then the DNA was labeled by streptavidin-functionalized QDs as shown in Figure 3. Nonviral vector chitosan, the backbone of which was labeled with Cy5, was mixed with the DNA, and the nanocomplexes were applied to HEK293 cells. The distribution and unpacking of the individual nanocomplexes within cells were successfully monitored by fluorescence


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Fig. 2. Typical design of spFRET for detecting protein–protein interactions. (A) The quantum dot (QD)–maltose-binding protein conjugates were prepared by dihydrolipoic acid (DHLA)-capped QD. (B) Experimental setup for spFRET and (C) detected signals from the setup. Only the signals above threshold indicated by arrows are recognized as “on.” The figures were reproduced with permission from Pons et al.29 spFRET = single particle fluorescence resonance energy transfer. LP = long pass. APDD = avalanche photodiode for donor. APDA = avalanche photodiode for acceptor. λexc = excitation wavelength.

microscopy at a single-particle level (Fig. 3). Making use of this QD-FRET technique, rational design of more efficient nonviral vectors will be possible in the nonviral GDS. Although QD-FRET will provide a new insight in the study of DDS and GDS, it is still a primitive field; thus, cytotoxicity or size effect of QD itself should be deeply studied for further development of this technique.


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Fig. 3. A nanocomplex designed for monitoring the behavior within cells. (A) Plasmid DNA was labeled with quantum dot (QD) through PNA–DNA binding. DNA was condensed by chitosan through electrostatic interactions, and nanocomplexes were formed as shown in (B) the transmission electron microscope (TEM) image. The fluorescent images of (C) 605QD-labelled DNA, (D) the nanocomplexes, and (E) disrupted nanocomplexes. Single nanocomplex showed sufficient fluorescence resonance energy transfer (FRET) signal for tracking the motion in cells. The figures were reproduced with permission from Ho et al.38

Cytotoxicity Nanoparticles, which have engineered structures with the size range of 100 nanometers or less, are now commercially

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available and spread to all parts of daily life such as cosmetics and drug carriers. While these expansions of nanomaterials market, their intrinsic toxicity is still unknown and sciencebased elucidation of the cytotoxicity of nanomaterials is strongly expected. From a toxicological perspective, particle size and surface area are important material characteristics. As the size of a particle decreases, its surface area increases and the ratio of the constituent atoms or molecules facing the surface gets greater39 (Fig. 4). The increase in surface area determines the potential number of reactive species on the particle surface. For catalyst use, this change in the physicochemical and structural properties generally leads to high efficiency in spite of the same weight of materials. However, from the viewpoint of toxicity, such highly reactive materials may have potential adverse effect in organisms. Not only the large surface area but also the size of nanomaterials may be a key factor to determine cell permeability. In this section, cytotoxicity of QDs will be reviewed from the viewpoint of particle size and surface coating. Cytotoxicity of QDs has become a serious problem especially for tissue engineering in which a reliable method for stem cell tracking is of key importance. Seleverstov et al. compared the cytotoxicity and intracellular processing of two differentsized peptide-conjugated QDs in human mesenchymal stem cells.40 They used commercially available QDs, QD525 and QD605, which have identical chemical components but differ in their sizes. QD525 and QD605 used here is spherical in shape with a diameter of 4 nm and a spheroidal shape of 12 nm in the major axis and 5 nm in the minor axis, respectively. Cellular uptake of both QD types has no significant differences after 24 h of post labeling. In 72 h cultures, however, the majority of cells presented that the cytoplasms are free from only QD525. To understand the detail of this phenomenon, they performed TEM observation and found a significant number of autophagosomes, swollen mitochondria,

Fig. 4. Percent surface molecules as a function of particle size. Surface molecules increase exponentially when particle size decreases below 100 nm. This characteristic of nanoparticles results in both positive and negative effects. The figure was reproduced with permission from Seleverstov et al.40

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and Golgi cisterns. Approximately 2% of the cells exhibited features of autophagic cell death (Fig. 5). Some QD525 aggregates in the nuclei were also observed, in which QD605 did not localize. This size-dependent uptake and activation of autophagy by QDs should be carefully considered especially for stem cell tracking, which requires nontoxic, multicolored, simultaneous, and long-time tracking. Lovric´ et al. also pointed out size-dependent uptake of QD.41 Small uncoated CdTe QDs with a diameter of 2.2 nm were more toxic in PC12 and N9 cells than the large one with a diameter of 5.2 nm. Size and charge of QDs affected their subcellular localization and cytotoxicity which was characterized by chromatin condensation and membrane blebbing. At the same time, they also suggested that addition of bovine serum albumin (BSA) and N-acetylcysteine (NAC) significantly decreased cytotoxicity. In the case of CdSe, a similar study was reported that BSA coating contributed to maintain hepatocyte viability. Along with the size effect of QDs, biocompatible surface coatings are essential in the biological application of QDs to prevent intrinsic toxicity of QD constituent such as Cd. The most likely passway is thought that QD-induced toxicity is correlated with the liberation of free cadmium ions (Cd2+), i.e, surface oxidation by UV irradiation triggers breakage of shells

Fig. 5. TEM image of autophagic cell death of human mesenchymal stem cells after 72 h labeling by QD525. Almost all nanoparticles are included in big vacuoles indicated by the black arrow, and the cytoplasm was totally destructed with big vacuoles. The figure was reproduced with permission from Oberdorster et al.39


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and surface coatings, and subsequent release of free Cd2+ causes lethal damage to the cell.42,43 CdTe QDs are also suggested that the induction of cell death via mechanisms involving both Cd2+ and reactive oxygen species accompanied by lysosomal enlargement and intracellular redistribution.44 Chang et al. scrutinized whether cellular toxicity arises from the interaction of QD surface molecules with cell membranes or from the intracellular uptake of endocytosed QDs, which is influenced by surface molecules on QDs.45 They concluded that improved biocompatibility arises from the minimized intracellular uptake of QDs by endocytosis. Since endosomal pH is generally lower than 6, this uptake could easily break the shell without surface-coating modifications. Even at high concentrations of polyethylene glycol (PEG)-substituted QDs in extracellular medium (5– 20 nM), it showed minimal cytotoxicity. On the other hand, high intracellular level of QDs showed significant cytotoxicity which might have arisen from the breakdown of endocytosed QDs by cellular degrative mechanisms. Avoiding endocytic uptake of QDs by improved biocompatible surface modifications is an important issue to expand QD applications to biological field without any concerns. Derfus et al. also reported that endocytosis of PEG-substituted QDs with cationic liposomes results in colocalization of the majority of the QDs in the endolysosomal compartment.46 They studied both biochemical methods using cationic liposomes and physical methods using electroporation and microinjection to characterize delivery of QDs into live cells. The former QDs/cationic liposome complexes and electroporation were efficient to deliver QDs to the cytoplasm, yet QDs tend to form large aggregates that can restrict subsequent trafficking into the nucleus. In contrast, although microinjection requires each cell to be individually manipulated, microinjection delivers QDs to the cell interior in a monodispersed form. The influence of the formation of aggregates in cells through biochemical methods is not well understood so prompt evaluation of the cytotoxicity of the formation of QD aggregates in the cytoplasm is required.

approach including surface coatings, cell types, and injection methods. Not only QDs but also other nanoscaled materials should exploit their positive aspects and avoid potential toxic effects with a multidisciplinary cooperation involving toxicologists, medical experts, molecular biologists, and materials scientists.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

Conclusions The introduction of QDs for biomedical applications opens numerous opportunities for optical imaging and diagnostics, and so forth. Clearly, QDs will provide new insights into the technique on the basis of single-molecule detection. Their bright and photostable characteristics, which are almost beyond expectation, enable molecular motions to be traced over a few tens of minutes. Based on FRET, biochemical reactions and conformational changes occurring in the surface vicinity could be detected at a single-particle level. Combining these unique characters of QDs and sophisticated organic fluorophores, novel nanohybrids or concepts are expected. One urgent task in bio-applications of QDs is to establish an evaluation


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