Home
Search
Collections
Journals
About
Contact us
My IOPscience
Labelling of cells with quantum dots
This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2005 Nanotechnology 16 R9 (http://iopscience.iop.org/0957-4484/16/2/R01) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 128.42.202.150 The article was downloaded on 03/09/2012 at 23:25
Please note that terms and conditions apply.
INSTITUTE OF PHYSICS PUBLISHING
NANOTECHNOLOGY
Nanotechnology 16 (2005) R9–R25
doi:10.1088/0957-4484/16/2/R01
TOPICAL REVIEW
Labelling of cells with quantum dots Wolfgang J Parak1,3 , Teresa Pellegrino1 and Christian Plank2 1 Center for Nanoscience, Ludwig Maximilians Universit¨at M¨unchen, Amalienstrasse 54, 80799 M¨unchen, Germany 2 Institute of Experimental Oncology, Technische Universit¨at M¨unchen, Ismaninger Strasse 22, 81675 M¨unchen, Germany
E-mail:
[email protected] Received 2 September 2004, in final form 15 December 2004 Published 25 January 2005 Online at stacks.iop.org/Nano/16/R9 Abstract Colloidal quantum dots are semiconductor nanocrystals well dispersed in a solvent. The optical properties of quantum dots, in particular the wavelength of their fluorescence, depend strongly on their size. Because of their reduced tendency to photobleach, colloidal quantum dots are interesting fluorescence probes for all types of labelling studies. In this review we will give an overview on how quantum dots have been used so far in cell biology. In particular we will discuss the biologically relevant properties of quantum dots and focus on four topics: labelling of cellular structures and receptors with quantum dots, incorporation of quantum dots by living cells, tracking the path and the fate of individual cells using quantum dot labels, and quantum dots as contrast agents.
1. Introduction To investigate cells and cellular processes it is very important to visualize structures and compartments within cells and molecules that are involved in the cell’s biochemistry. Since cells are almost transparent to visible light and individual molecules are very small, typically with microscopy no direct observation of structural compartments or molecules is possible. Therefore the structures or molecules of interest have to be labelled with a marker that can be directly observed. One of the most common techniques used in cell biology is certainly fluorescence labelling [1–3]. Either fluorophores can be directly attached to the target to be visualized, or they can be attached to a molecule (e.g. an antibody) [4] that binds to the target via molecular recognition. Such fluorescence labelling techniques are well established, and are used for most cellular labelling experiments. However, traditional fluorophores have their limitations. The conformation of fluorophores is very sensitive to their local environment, e.g. to thermal fluctuation of the solvent [5]. As a consequence of conformational fluctuations, fluorophores can be reversibly transferred to states in which they cannot fluoresce anymore 3 Author to whom any correspondence should be addressed.
0957-4484/05/020009+17$30.00 © 2005 IOP Publishing Ltd
and the fluorescence of single molecules statistically goes ‘on’ and ‘off’, which is called blinking [6]. Upon optical excitation, organic fluorophores can undergo irreversible lightinduced reactions such as photo-oxidation. These molecules are then no longer fluorescent, a phenomenon known as photobleaching [7]. Photobleaching limits the time for which such labels can be observed under a fluorescence microscope. This limitation is of special importance for all studies in which the label is to be observed over extended periods of time, as is the case in any experiment involving temporally resolved imaging. Another restriction of organic fluorophores is the inherent resolution limit of optical microscopy. Although the fluorescence of individual fluorophores of a few nanometre sizes can be observed, such single-molecule fluorescence techniques are based on diluted distributions of labels, i.e. the distance between individual fluorophores has to be bigger than the resolution limit. Therefore, with classical wide field optical microscopy, no structures smaller than a few hundred nanometres can be resolved. To overcome some of the above described limitations there is an ongoing development of alternative techniques. One established technique is the use of colloidal metal particles, in particular small gold particles. These labels can be almost as small as organic fluorophores, but due to their metallic
Printed in the UK
R9
Topical Review
nature they offer enough contrast to be imaged with electron microscopy. Since the resolution for electron microscopy is much better than for optical microscopy, biological structures can be visualized with nanometre resolution [8– 10], for example the protein distribution in membranes. Unfortunately, TEM is typically only possible with fixed and thus dead samples. However, many interesting labelling experiments have also been reported in which gold colloids have been imaged with optical microscopy [11]. Using gold particles instead of fluorophores circumvents the drawbacks of photobleaching and blinking. In the last decade semiconductor nanocrystals (so-called quantum dots) have been introduced as a new type of colloids for biolabelling. The fluorescence wavelengths of these nanoparticles are size dependent, as will be discussed later. First biological applications have been discussed in many news and views/feature articles [12–18] and reviews [19–28]. Since these fluorescent nanoparticles are inorganic solids, they can be expected to be more robust than organic fluorophores (e.g. towards photobleaching) and in addition they can also be observed with high resolution by electron microscopy. The use of nanoparticles in biology and medical applications is a multi-disciplinary field of science where aspects of synthetic chemistry, physics and biology are equally important. It is difficult to find common language and thinking among these scientific disciplines. Chemists and physicists have produced and characterized in detail a multitude of high quality colloidal particles with tuneable properties. However, their application in life sciences has often been restricted to demonstration experiments despite their potential usefulness in a wide variety of applications. Here we want to review the use of semiconductor nanocrystals as fluorescence labels emphasizing chemical and physical aspects of these materials as the basis of their applicability in biological sciences. We will discuss how these particles have been used in experiments with cells and living tissue and we will highlight some of their potential future applications in the life sciences.
2. Chemical and physical properties of quantum dots relevant for their use as fluorescence labels in cell biology 2.1. Solubility in aqueous buffers High quality nanocrystals (in terms of crystallinity and size distribution) of various semiconductor materials such as CdS, CdSe, CdTe, or CdSe/ZnS can be synthesized by a variety of approaches [29–32]. Most commonly the synthesis is carried out in organic solvents at high temperatures in the presence of surfactants, these reaction conditions being required to yield monodisperse and stable particles. This synthetic approach produces surfactant-coated particles, the polar surfactant head group attached to the inorganic surface, the hydrophobic chain protruding into the organic solvent, mediating colloidal stability. At this stage, the particles will be well dispersed in solvents such as toluene or chloroform, but because of their hydrophobic surface layer they are not soluble in aqueous media. However, almost all experiments involving cells require water-soluble materials. This problem has been solved by replacing the surfactant layer or by R10
coating with an additional layer introducing either electric charge or hydrophilic polymers for mediating solubility in water. Coulomb repulsion between nanocrystals with surface charge of the same polarity prevents aggregation in water. However, in salt-containing solutions such as cell culture media charged particles tend to aggregate, a phenomenon well known in colloid chemistry (‘salt-induced aggregation’). Qualitatively, this phenomenon is explained by the reduction of the thickness of the so-called diffuse layer of a counter-ion cloud surrounding a charged particle with increasing ionic strength (the thickness of the diffuse layer is inversely proportional to the Debye–H¨uckel parameter which is proportional to the square root of the ionic strength). As a consequence, in salt-containing solutions charged particles can approach each other closely enough during thermal collisions such that the repulsive electrostatic forces are overcome by the short range attractive van der Waals forces. The system flocculates. The second principle, using hydrophilic polymers, involves steric stabilization. The considerable space occupied by a hydrophilic or amphipathic coating polymer such as polyethyleneglycol [33] or dextrane [34] cannot be occupied by another polymer chain from the coating layer of another particle. The particles then cannot approach each other such that van der Waals forces lead to flocculation. In any case, both discussed principles involve the conversion of hydrophobic to hydrophilic surfaces [23] (see figure 1(a)). In practice, hydrophobic nanocrystals can be made hydrophilic by several methods. Most of them rely on the exchange of the hydrophobic surfactant coatings with ligand molecules that carry on one end functional groups that are reactive towards the nanocrystal surface, and hydrophilic groups on the other end, which ensure water solubility. The most frequently used anchoring groups reactive to the surface of semiconductor nanocrystals are thiol (–SH) functionalities, and carboxyl (–COOH) functionalities are most often used as hydrophilic head groups. For pH values >5 the carboxyl groups are deprotonated and the nanocrystals repel each other by the negative charge of the carboxylate ions (–COO− ). Examples for such mercaptohydrocarbonic acids (SH– · · ·–COOH) are mercaptoacetic acid [35–38], mercaptopropionic acid [39], mercaptoundecanoic acid [40], mercaptobenzoic acid [41], dihydrolipoic acid [42], and cysteine [43, 44]. Pinaud et al have used synthetic peptides with multiple cysteines as the anchor [45]. Also non-charged molecules like dithiothreitol [46], organic dendrons [47], and pyridine-functionalized polyethylene glycol (PEG) [48] have been used. In the case of pyridine-functionalized PEG the pyridine functionality is used to bind to the nanocrystal surface, whereas the hydrophilic PEG chain stabilizes the nanocrystals in aqueous solution by steric repulsion. Although ligand exchange from hydrophobic to hydrophilic surfactants is straightforward, there are drawbacks connected with this method. Unfortunately, the bond between thiol (–SH) groups and semiconductor surfaces is not very strong and therefore from the viewpoint of long term stability the hydrophilic ligand shell around the nanocrystal is prone to disintegration, which is tantamount to particle aggregation [49, 50]. An alternative approach is based on the growth of a hydrophilic silica shell around the nanocrystals, through a
Topical Review
b1)
a) c1)
b2)
c2)
100 nm
Figure 1. Schematic representation of the use of colloidal quantum dots for biological labelling. (a) Water-soluble quantum dots comprise a semiconducting core (drawn in grey) and a hydrophilic shell (drawn in black). (b) In order to specifically label biological structures water-soluble quantum dots have to be conjugated with biological molecules (drawn in red) such as antibodies. The biological molecules can be either adsorbed (b1) to the semiconducting core or bound to the hydrophilic shell (b2). (c) Bioconjugated quantum dots bind specifically to designated receptors. In (c1) the ligand (e.g. a primary antibody, drawn in red) bound to the quantum dot directly recognizes specific receptor molecules (drawn in blue). In (c2) the receptor (drawn in blue) is first labelled by a primary ligand (e.g. primary antibody, drawn in green), which in turn is recognized by the ligand (e.g. a secondary antibody, drawn in red) bound to the quantum dot. Inset: red fluorescent CdSe/ZnS quantum dots have been made water soluble by coating them with an amphiphilic polymer. The image shows a transmission electron micrograph (TEM) of some of these quantum dots adsorbed on a TEM grid.
process known as surface silanization [33, 50–55]. Similarly as described above, a first step involves a ligand exchange to substitute the original hydrophobic surfactant covering the nanocrystal surface. The most frequently used molecule for this step is mercaptopropyltrimethoxysilane. The mercapto (–SH) group binds to the semiconductor surface and three methoxy groups are exposed to the solvent. The advantage of this method is that the methoxysilane groups can react with each other under the formation of siloxane bonds (–Si–O–CH3 + CH3 –O–Si– + H2 O → –Si–O–Si– + 2CH3 OH). In this way the surfactant shell around the nanocrystal becomes cross-linked and has improved stability towards disintegration. To render the silica shell hydrophilic in a next step, molecules bearing methoxysilane groups at one and hydrophilic groups at the other end are incorporated in the shell again by the formation of siloxane bonds, resulting finally in a multi-layer shell. If methoxysilane molecules with phosphonate groups are added, the particle surface becomes negatively charged and particles repel each other by electrostatic interaction. If methoxysilane molecules with polyethyleneglycol groups are used, particles are stabilized by steric repulsion [33]. Silanized semiconductor nanocrystals are extremely stable in electrolytic solution, but the silanization process is somewhat laborious and the resulting shell inhomogeneous. Recently, a method to coat hydrophobic semiconductor nanocrystals with amphiphilic polymers has been reported [56–59] (see figure 1(a) and the inset). In this approach the hydrophobic tails of the polymer intercalate the hydrophobic surfactant molecules on the nanocrystal surface and thus form an additional coating. The water solubility of polymercoated nanocrystals is ensured by hydrophilic groups of the
polymer which self-assemble on the outside of the polymer shell. In order to further stabilize the polymer shell around the nanocrystal, the individual polymer chains are cross-linked. One advantage of this method is that it is not based on ligand exchange, i.e. not on replacing the original hydrophobic surfactant with new hydrophilic molecules, but the whole nanocrystal is covered with a cross-linked polymer shell. First results indicate that this shell is thinner and more homogeneous than silica shells although the wrapping of multiple shells around the inorganic core (surfactant, polymer, counter-ions etc) make the overall diameter larger than the corresponding nanocrystals transferred in water by surfactant exchange. Furthermore, hydrophobic semiconductor nanocrystals have also been stabilized in aqueous solution by embedding them in phospholipid micelles [60]. As can be seen from this short summary, several reliable methods exist to stabilize nanocrystals in aqueous solution. Although there is no optimum protocol available yet which includes all the advantages of the individual procedures, state of the art nanocrystals have reached a degree of performance regarding water solubility that is sufficient for biological experiments. One conceptual advantage of fluorescent colloidal nanocrystals over organic fluorophores is evident. Organic fluorophores with different colours of fluorescence are typically just different molecules and thus have different surface properties. On the other hand, the optical properties of water-soluble nanocrystals of different colours of fluorescence are determined by the material and the size of the semiconductor nanocrystal, but their surface is defined by the hydrophilic coating in which the nanocrystals are embedded. In other words, the optical properties of colloidal nanocrystals R11
Topical Review
b)
a)
1.5
Absorption: fluorescein TAMRA Cy5
Absorption/ Fluorescence
0.8
Fluorescence: fluorescein TAMRA Cy5
0.6
0.4
Polymer coated CdSe/ZnS
Absorption / Fluorescence
1.0
Absorption: green orange red
1.0
Fluorescence: green orange red
0.5
0.2
0.0 400
500
600
700
800
wavelength (nm)
0.0 400
500
600
700
800
wavelength (nm)
Figure 2. Comparison of absorption (solid curve) and fluorescence (dashed curve) spectra of organic fluorophores and colloidal quantum dots. (a) Normalized absorption and fluorescence spectra in water of three typical organic fluorophores (fluorescein, tetramethylrhodamine (TAMRA), and Cy5). (b) Normalized absorption and fluorescence spectra in water of polymer-coated CdSe/ZnS quantum dots of different size.
are defined by the semiconductor core inside, but their surface chemistry is defined by the outside of the chemical coating, which can be identical for all different types of nanocrystals.
(ii) an (optional) shell of a semiconductor material with higher bandgap than the material of the core (e.g. a ZnS shell around CdSe cores) to increase the quantum yield, and (iii) a hydrophilic coating to warrant water solubility.
2.2. Photo-physical properties
The absorption and fluorescence spectra of quantum dots are remarkably different from those of organic fluorophores (see figure 2). Organic fluorophores like fluorescein, rhodamine, or Cy-dyes typically can only be optically excited within a narrow window of wavelengths, the spectral position of which depends on the particular fluorophore. The fluorescence emission is also limited to a certain window of wavelengths. Remarkably, the fluorescence spectra are not symmetric, but exhibit a tail to longer wavelengths called the ‘red tail’. Colloidal quantum dots on the other hand have a continuous broad absorption spectrum. Fluorescence can be excited with any wavelength shorter than the wavelength of fluorescence. Therefore many quantum dots with different colours of fluorescence can be excited with one single wavelength (light source), in contrast to organic fluorophores, which require a certain wavelength for every different fluorophore and thus colour of fluorescence. The flexibility to choose any wavelength shorter than the wavelength of fluorescence also helps to reduce auto-fluorescence of biological samples, simply by selecting the most appropriate excitation wavelength for which auto-fluorescence is minimum. Furthermore, the possible large separation between excitation and emission wavelength allows for collecting broad frequency windows in the emission spectra, which results in improved sensitivity of detection, whereas for typical organic fluorophores the emission spectra have to be recorded in a narrow spectral window in order to avoid overlap with the excitation. The fluorescence emission spectra of quantum dots are relatively narrow, symmetric and do not exhibit a red tail. As a consequence many different colours can be distinguished without spectral overlap. Since each colour can code for one channel of information this is
Semiconductor nanocrystals can be physically described as quantum dots [61]. They exhibit atom-like size-dependent energy states due to the confinement of the charge carriers (electrons, holes) in three dimensions [62–65]. As a consequence the energy gap (i.e. the energy difference between the excited and the ground state) of a quantum dot strongly depends on its size [61]: the smaller the diameter of the quantum dot is, the bigger the energy gap becomes. Upon optical excitation, electron–hole pairs are generated, i.e. electrons are excited from the valence to the conduction band, which is analogous to exciting electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) in organic fluorophores. The recombination of electron–hole pairs, i.e. relaxation of the excited state, results in the emission of fluorescence light. The wavelength of fluorescence depends on the bandgap and thus on the size of the quantum dot [66–69]. Small CdSe nanocrystals (about 2 nm diameter) for example are fluorescent in the green, whereas bigger ones (about 5 nm diameter) are fluorescent in the red. In first order, the colour of fluorescence is only determined by the diameter of the semiconductor core. To reduce non-radiative relaxation on the particle surface, which is equivalent to recombination of electron–hole pairs without emitting fluorescence light, a second shell of a semiconductor material with higher bandgap can be grown around the semiconductor core [29]. Therefore typical water soluble nanocrystals comprise (i) a semiconductor core (CdSe, CdS, CdTe), the diameter of which determines the wavelength of fluorescence, R12
Topical Review
important for all experiments in which different labels have to be used in parallel. For biological fluorescence labelling this means that the more colours can be resolved, the more different compartments/structures/processes can be labelled simultaneously, each with a different colour. High quality multi-colour labelling of different cellular structures has been demonstrated for example by Wu et al [56] and by Mattheakis et al [70]. Even higher degrees of signal multiplexing can be obtained if several quantum dots of different colours are connected to one entity, as for example in the case of quantum dot decorated microspheres. Each microsphere with a different ratio of quantum dots of different colours represents one spectral code [71, 72]. The group of Shuming Nie has embedded quantum dots of six different fluorescence colours with ten intensity levels in polystyrene microspheres (i.e. varying the number of quantum dots of each colour per microsphere in ten steps). They demonstrated that these microspheres can be used to tag spectral codes on biological molecules and that in theory these microspheres can be used to code one million nucleic acid or protein sequences [72]. Maybe the biggest advantage of quantum dots is their reduced tendency to photobleach. Organic fluorophores can undergo irreversible light-induced reactions upon optical excitation, which result in loss of fluorescence. Due to their inorganic nature quantum dots suffer much less from photobleaching. This has been demonstrated in many practical labelling experiments in which the performance of quantum dots has been compared with fluorophores typically used in cell biology [35, 40, 42, 43, 56, 60, 73–75]. It has been reported that upon optical excitation the fluorescence of (water soluble) quantum dots even increases at the beginning, a phenomenon known as photo-brightening [38, 50, 76, 77]. Reduced photobleaching is of particular importance for experiments which involve long term imaging, e.g. for any time-resolved study, such as fluorescence labelling of transport processes in cells, or tracking the path of single membrane-bound molecules [78]. Another example is experiments in which adjacent areas have to be excited over extended periods of time, as is the case for obtaining high magnification fluorescence image z-stacks for threedimensional reconstructions of cell or tissue compartments. Unfortunately, colloidal quantum dots suffer from blinking, very similar to organic fluorophores [19, 79–83]. However, in a recent study it was speculated that blinking of colloidal quantum dots might be due to the fact that in typical experiments quantum dots are in the proximity of surfaces, whereas there is experimental evidence that no blinking might occur for quantum dots freely suspended in solution far from any surface [84]. Besides spherical nanocrystals asymmetric nanorods can also be synthesized. Due to their anisotropic shape their emitted fluorescence light is polarized [85]. This potentially enables the detection of the orientation of labelled structures. However, no biological applications have been reported so far that make use of this fact. Also fluorescence resonance energy transfer (FRET) involving colloidal quantum dots has been demonstrated [86–89]. Finally colloidal quantum dots have been also used as contrast agents for optical recordings of tissue compartments inside animals [84, 90]. Another interesting feature of colloidal quantum dots is their long fluorescence lifetime [91]. The fluorescence lifetime
of typical organic fluorophores lies in the range of a few nanoseconds, which matches with the decay time of autofluorescence of many biological samples. Colloidal quantum dots on the other hand can have fluorescence lifetimes of a few tens of nanoseconds [91]. If time-gated detection is employed, i.e. fluorescence is recorded a few nanoseconds after optical excitation with a light pulse, then most of the auto-fluorescence background is already decayed, which results in an improved signal-to-noise ratio. This technique has been demonstrated for colloidal CdSe/ZnS quantum dots inside fibroblast [91]. Colloidal quantum dots also have a significant two-photon cross section, which is up to two to three orders of magnitude higher than that of typical organic fluorophores [84]. Multiphoton excitation is especially suited for thick specimens because it allows for imaging of structures deep inside biological tissues. In this way colloidal CdSe/ZnS quantum dots have been used for example as contrast agents for imaging of blood vessels in living mice [84]. Also quantum dots inside individual cells have been imaged with two-photon microscopy [36, 74]. It has to be pointed out that the fluorescence of quantum dots decreases when originally hydrophobic particles are transferred from organic into aqueous solution by giving them a hydrophilic surface coating as described in the above section. In other words, the quantum yield of hydrophilic quantum dots in aqueous solution is lower than that of hydrophobic quantum dots in organic solution. However, they retain their basic optical properties, such as the shape of the absorption and emission spectra, and reduced photobleaching. It is evident that colloidal quantum dots have different optical properties than organic fluorophores and therefore they are superior for certain types of experiments. Because of their narrow excitation, broad absorption, and reduced photobleaching, quantum dots seem to be especially promising for all labelling experiments that involve the detection of many colours of fluorescence over extended periods of time. 2.3. Conjugation of quantum dots with biological molecules Biological molecules can be linked to the surface of colloidal quantum dots in order to introduce specific functionalities [20]. Stated from another perspective, biological molecules can be fluorescence labelled by attaching quantum dots. In general there are two strategies to conjugate quantum dots with biological molecules (figure 1(b)). First, biological molecules can be functionalized with a chemical group that is reactive towards the semiconductor surface, for example mercapto (– SH) groups (figure 1(b1)). Thiols (–SH) bind to the surface of the most often used semiconductor materials (CdSe, CdS, CdTe, ZnS) and therefore quantum dots can be conjugated to biological molecules bearing mercapto (–SH) groups in this way [92, 93]. If colloidal quantum dots are incubated in a solution of such molecules some of the surfactant molecules on the nanocrystal surface will be replaced by these molecules. This method is basically a partial ligand exchange in which part of the hydrophilic surfactant that stabilizes the nanocrystal in aqueous solution is replaced by biological molecules. One of the problems related to ligand exchange is the stability of the resulting nanoparticle–biomolecule conjugates, since the bond between mercapto (–SH) groups and semiconductor surfaces is R13
Topical Review
not very strong. To overcome this problem, multiple mercapto groups can be used as anchor, as has been demonstrated for example by conjugating quantum dots with peptides that had multiple cysteine residues on one end [45]. In an alternative method biological molecules are covalently linked to the quantum dot surface on their outer hydrophilic shell (figure 1(b2)). Most simply, biological molecules can be adsorbed to the hydrophilic shell in a nonspecific way [40, 94]. In a more specific way, molecules can be adsorbed to the hydrophilic shell by electrostatic interaction. Most colloidal quantum dots are negatively charged and therefore positively charged biological molecules stick to their surface. In the case of proteins it is also possible to genetically engineer a positive zipper to one end of the protein, with which the protein then is electrostatically adsorbed to the quantum dot surface [42, 95– 97]. Finally, biological molecules can also be covalently attached to the quantum dots by means of cross-linker molecules [20, 33, 35–38, 43, 46, 52, 56, 60]. This requires hydrophilic surfactant shells with reactive groups, such as –COOH, –NH2 , or –SH. Since most biological molecules bear these functional groups or can be easily modified with them, a covalent linkage between functional groups on the quantum dot surface and functional groups of the biological molecule can be formed. Typical procedures include carbodiimide activation of carboxylic acid groups on one entity followed by amide bond formation to amino groups on the other entity or the linkage of thiol and amino groups using bifunctional cross-linkers such as SMCC (4-(N -maleimidomethyl)cyclohexane-1-carboxylic acid N -hydroxysuccinimide ester) [98]. A huge variety of suitable cross-linking reactions can be found in textbooks [98]. Conjugation of quantum dots with biological molecules is well established and has been demonstrated for a whole variety of molecules, including small molecules like biotin [52], folic acid [20], or the neurotransmitter serotonin [93], peptides [37, 92], proteins like avidin or streptavidin [42, 56], albumin [40], transferrin [35], trichosanthin [36], the lectin wheat germ agglutinin [38], or antibodies [37, 42, 43, 56, 96], and DNA [33, 46, 55, 60, 99, 100]. Instead of directly conjugating biological molecules to the hydrophilic outer shell of quantum dots they can also be attached via the streptavidin–biotin system. For this purpose streptavidin is covalently bound to the surface of the quantum dots as described above. These quantum dots are now reactive towards biological molecules that are bearing a biotin moiety. This approach is very universal, because many biological molecules are available with biotin tags or can easily be synthesized. Streptavidin-coated quantum dots are commercially available and can be readily conjugated with biotinylated proteins and antibodies [56, 73, 75, 78, 101]. 2.4. Preservation of biological activity of molecules conjugated to quantum dots Biological molecules can be conjugated to colloidal quantum dots as described above. For potential applications it is important to know if the functionality of the molecules is retained or whether it is impaired by the quantum dot. Since colloidal quantum dots have the size of a few nanometres, the attachment of such objects to a biological R14
molecule might well influence its properties, e.g. in terms of steric hindrance. It is known for example that the conjugation of single-stranded oligonucleotides to the surface of gold nanoparticles reduces their ability to hybridize with complementary oligonucleotides [102]. Therefore, preservation of the functionality of the molecule cannot be taken for granted and must be proven experimentally. Functionality of biological molecules can be regarded from different perspectives. One main idea of applying quantum dots in cell biology is their use as fluorescence markers to label structures/compartments/molecules in cells. For this purpose quantum dots are conjugated to ligand molecules (such as antibodies) which recognize specific receptors, i.e. the structure/compartment/molecule which is to be labelled. Therefore, it is important that the quantum dots do not interfere with molecular recognition. In other words, the quantum-dot-labelled ligand molecule must still be able to find its target. In basically all reported experiments the attachment of quantum dots to biological ligands did not affect their binding ability to the corresponding receptor. For example, the hybridization of oligonucleotides conjugated to quantum dots to complementary oligonucleotides [46, 60, 99, 100], the binding of biotin/avidin quantum dots conjugated to avidin/biotin [42, 52, 56, 73, 75, 78, 101], and the specific targeting of peptides [37, 92], proteins [35, 38], and antibodies [37, 42, 56] to their receptors conjugated to quantum dots have been demonstrated. However, the preservation of molecular recognition is just one aspect of functionality. Often biological molecules have certain effects (for example enzymes can catalyse reactions), or cause certain actions (such as neurotransmitters opening or blocking ion channels). Some studies are available at present in which the functionality of biological molecules labelled with colloidal quantum dots is investigated. Zhang et al claim that the enzymatic activity of the ribosome-inactivating protein trichosanthin did not change significantly when it was attached to colloidal CdSe/ZnS quantum dots [36]. Dahan et al report that labelling of membrane-bound receptors with quantum dots did not have drastic effects on the diffusion behaviour of the receptors in membranes [91]. Kloepfer et al have investigated the effect of quantum dot conjugation on the functionality of the protein transferrin and conclude from three experiments that the protein’s function is not compromised by its attachment to quantum dots [38]. In a detailed study by the Rosenthal group the effect of quantum dot conjugation to the neurotransmitter serotonin was investigated [93]. Their experimental data suggest that conjugation to quantum dots dramatically reduces the binding affinity of serotonin to serotonin-transporter proteins. Moreover, for Xenopus oocytes expressing ionotropic serotonin receptors a serotonin-induced inward current was recorded for perfusion with free serotonin, but no current was observed in the case of perfusion with serotonin–quantum dot conjugates. The experiments from the Rosenthal group clearly demonstrate that the conjugation of biological molecules to quantum dots can impair their functionality. Although some possible reasons such as steric hindrance by the volume of the nanoparticle have been suggested in a speculative way the detailed effects remain to be investigated. We can conclude that in many cases the function of biological molecules is not drastically changed upon
Topical Review
conjugation to colloidal quantum dots. In particular, the ability of molecular recognition, i.e. the affinity of ligands to their receptor, seems at least to be partially preserved. On the other hand it has been reported that sophisticated molecules that trigger an effect, like the opening of an ion channel, can be impaired upon conjugation to quantum dots. However, to our knowledge there are no detailed studies available at present in which the functionality of molecules conjugated to quantum dots is analysed in a quantitative way, e.g. in terms of binding constants or catalytic activity. 2.5. Toxicity/biocompatibility Since constituents of colloidal quantum dots such as cadmium [103] or selenium [104] are toxic to many cells, harmful effects can be expected, especially when the hydrophilic shell around the quantum dots is not stable and they might dissolve under the release of toxic ions. On the other hand, some cells have developed mechanisms to cope with such ions, such as assembling them to particles in a biomineralization process, which efficiently removes ions from solution. In this way, fluorescent colloidal CdS [105, 106] or PbS [107] quantum dots are grown in some cells. In terms of biocompatibility or toxicity we have to differentiate between several modes how quantum dots are introduced into cells. Certainly the results will also strongly depend on the type of cells and on the hydrophilic shell used to stabilize the quantum dots in aqueous solution. Quantum dots injected into the tail veins of mice are reported to cause no visible toxic effect after 24 h [92] and even after days [59, 84] (20 pmol quantum dots injected per gram animal weight [59]). Dubertret et al report the injection of quantum dots in individual cells of early stage Xenopus embryos [60]. Such embryonic systems are very sensitive to any perturbation and are therefore ideal for investigating toxic effects, which typically result in phenotype abnormalities. Whereas typical injections of 2 × 109 quantum dots per cell did not statistically change the health of the embryos, for injections of >5 × 109 quantum dots per cell abnormalities like changes in cell size, cell movement, axis elongation, and posterior truncations became apparent. Dubtret et al speculate that these abnormalities might be caused by changes in the osmotic equilibrium of the cells due to the injected quantum dots. Colloid quantum dots dissolved in the culture medium are ingested by many cells. This will be discussed in detail below. Concerning toxicity, some groups report as a general statement (e.g. without details about the concentration of quantum dots) that there are no detectable differences from unlabelled cells up to 2 weeks after incubation [45, 70, 74, 91]. Derfus et al measured the liver specific function of hepatocyte cells incubated with quantum dots in terms of the albumin secretion and could not determine any difference from untreated cells [108]. In a more quantitative way Hanaki et al demonstrated that the viability of cells was not affected for quantum dot concentrations of 0.4 g l−1 in the medium [40]. Jaiswal et al report that 400–600 nM quantum dots in the medium had no detectable effects on cell morphology or physiology [42]. Furthermore quantum dots in these concentrations did not interfere with the initiation
of development of Dictyostelium discoideum cells and their response to cAMP [42]. Finally, Winter et al report that the addition of 0.03 nM colloidal quantum dots (which were not ingested by the cells) did not alter the proliferation and attachment of cells within 5 days [37]. All studies described above can at best be called semiquantitative and most of them are based on judging the viability of the cells by optical light microscopy. In a quantitative way, cell viability can be measured with colorimetric assays, such as the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide). Such quantitative assays allow for measuring absolute numbers for the survival rate of cells upon incubation with toxins. With the MTT assay Mattheakis et al demonstrated that no toxicity of highly purified polymercoated CdSe/ZnS quantum dots could be observed for typical labelling concentrations of 10 nM [70]. Furthermore, the same authors demonstrated in a quantitative way for several cell lines that under the above mentioned conditions there is no interference of the quantum dot labels with the following cellular functions: ligand binding to cellular compartments, protein trafficking, and signal transduction. The same MTT assay has been used for the so far most comprehensive cytotoxicity study on quantum dots by Derfus et al [108]. For enhanced sensitivity this study was performed on primary hepatocyte cells from rats instead of using cell lines, since it is known that even low levels of Cd2+ reduce the viability of hepatocytes in vitro [109]. The authors have performed tests with different types of quantum dots. As a ‘worst case’, plain CdSe quantum dots coated with mercaptoacetic acid were used. In particular after UV exposure or exposure to air significant levels of free Cd2+ ions were found in the quantum dot solutions. The viability of the cells incubated with these samples correlated with the concentration of free Cd2+ ions in solution [108]. Based on these results the authors conclude that cytotoxicity of CdSe quantum dots correlates with the liberation of free Cd2+ ions from the CdSe lattice. Exposure to air induces oxidation of the quantum dot surface [29] and exposure to UV radiation catalyses oxidation of the quantum dot surface [49], which finally results in the release of Cd2+ ions. Free Cd2+ ions are known to interfere with the function of the mitochondria [110]. Derfus et al could demonstrate that release of Cd2+ ions und thus cytotoxicity can be reduced by coating CdSe quantum dots with appropriate shells [108]. Already the overcoating of CdSe quantum dots with a ZnS shell dramatically reduced Cd2+ release and cytotoxicity. Adsorbed bovine serum albumin (BSA) or coating the particles with a polymer shell further suppressed Cd2+ release. For these protected particles no cytotoxic effects were found for particle concentrations up to 1 mg ml−1 , which corresponds to µM concentrations. In summary, these results suggest that in the case of moderate concentrations of quantum dots toxic effects on cells can be neglected in first order, especially when Cd2+ release from the quantum dots is reduced by appropriate encapsulation of the CdSe cores. However, the field would still benefit from additional systematic studies on the concentration limits and on the dependence of the surface chemistry of the quantum dots. We also want to mention the following dilemma for labelling studies with animals or humans: cytotoxicity has certainly to be considered from a different perspective for labelling R15
Topical Review
cell cultures compared to labelling animals or humans. For cell cultures, inert quantum dots that do not release any toxic ions are perfectly suited. On the other hand, as will be described in detail later in this article, living cells ingest quantum dots: quantum dots can be used as contrast agents in animals and even usage in humans is thinkable. In the short range quantum dots as inert as possible are also desirable in order to prevent acute damage of the tissue. However, many of these ingested dots remain in the living tissue for months and presumably even for years [59]. No encapsulation can be as inert as to withstand degradation forever. This means that even quantum dots that have been encapsulated in several protection shells might slowly release toxic ions in the time course of years. Therefore, for studies on humans alternative quantum dot materials would be desirable, which would be based on biodegradable constituents. For magnetic resonance imaging on humans iron oxide nanoparticles have for example been used as contrast agents. These particles are also ingested by living cells [111], but they are deliberately not inert. Biodegradation of the particles finally results in free iron that is incorporated into haemoglobin [112] and after some months no residues of the particles remain in the body.
3. Labelling of cellular structures and receptors with quantum dots The most widespread method to visualize structures or certain molecules in cells is fluorescence microscopy. Due to their interesting optical properties colloidal quantum dots have been introduced as fluorescence labels for biological staining experiments since some years. Their application is in principle very similar to that of organic fluorophores. After attaching a ligand, for example an antibody, to the label [4], this conjugate binds with high specificity to its target receptor, which in turn can now be visualized by the fluorescence of the label (see figure 1(c)). Since the size of typical ligands is too big to allow their transport through natural pores in the cell membrane, fluorescence-labelled ligands have to be artificially introduced inside cells if structures in the cell’s inside are to be labelled (this is true both for organic fluorophores and for colloidal quantum dots). If only a few cells are to be labelled, fluorescence-labelled ligands can be injected into living cells with micropipettes [113, 114]. Also electroporation is used [115, 116]. In order to stain many cells in parallel, cells are typically fixed, i.e. stabilized by cross-linking their sugar skeleton on the cell surface (e.g. by glutaraldehyde) and their membrane is permeabilized with appropriate reagents (e.g. by triton). Fixed cells are dead and can be stored at 4 ◦ C. Fluorescence-labelled antibodies now can enter the cells by the created artificial pores. These labelling techniques are well established and protocols can be found in almost any textbook about cell biology [117, 118]. Using quantum dot labels is very similar to using their organic counterparts. Cells are fixed and permeabilized, incubated in buffer with ligand-conjugated quantum dots, for example oligonucleotide-conjugated quantum dots [46], rinsed, and fluorescence is recorded with an optical microscope. The first labelling experiment was performed in 1998 by the Alivisatos group [52]. Nowadays high quality images like the dual-colour labelling shown in figure 3(a) can R16
be obtained with standardized procedures. If antibodies are used as ligands, typically a two-step reaction is used. First, a primary antibody is reacted with the target. Then quantum dots conjugated with a secondary antibody, which targets the primary antibody, are added [43, 44, 56]. Since more than one secondary antibody can bind to each primary antibody, such cascade reactions amplify the fluorescence signal. However, the direct conjugation of antibodies to quantum dots is laborious; often biotinylated secondary antibodies are used, which are finally labelled by streptavidin–quantum dot conjugates via biotin–avidin interaction [73, 75, 119]. This strategy is advantageous, because it involves only one universal conjugation of quantum dots with streptavidin, and most antibodies are commercially available with an attached biotin group. Further signal amplification can be achieved with the so-called TSA enzyme amplification technique. Here the secondary antibody is conjugated with horseradish peroxidase (HRP) and quantum dots are conjugated to tyramide (e.g. via biotin–avidin interaction). Horseradish peroxidase converts tyramide to a very reactive oxidized intermediate, which rapidly binds to all proteins in close proximity. In this way the whole local environment of the secondary antibody is labelled with fluorescent quantum dots [73]. Quantum dot labelling of inner and outer structures of fixed cells has been used for a variety of receptors. If structures on the surface of cells are to be labelled, the quantum dot labels do not need to traverse the cell membrane. Therefore, for outside labelling no fixation and poration is required and labelling can also be performed with living cells [45, 56, 78, 93, 101]. In this way even the dynamic arrangement of single proteins within the cell membrane could be traced [78, 101]. Two examples for quantum dot labelling of membrane bound proteins, one from the Rosenthal group, the other from Quantum Dot Corporation, are shown in figure 3. Quantum dot labelling of cells is at present used by more and more groups and many new studies are to be expected in the future. We think that quantum dots will be used in the future in particular for time-resolved studies of single molecules, as demonstrated by Dahan et al and Lidke et al [78, 101]. The use of colloidal quantum dots in these experiments offered significant advantages compared to other methods: whereas single-molecule spectroscopy using conventional organic fluorophores as labels suffered from photobleaching which resulted in restricted observation times [120], the use of latex beads as labels suffered from the extended size of the beads [121]. It is likely that colloidal quantum dots never will completely replace organic fluorophores as fluorescence labels, but there are niches as long term imaging for which their advantages are obvious and therefore widely spread use is to be expected.
4. Incorporation of quantum dots by living cells In addition to the use of colloidal quantum dots as fluorescence markers to label cellular structures [52] the Nie group reported that quantum dots are incorporated by living cells [35]. Since then many groups have reported the uptake of quantum dots by individual cells or by animal tissue. The uptake of colloidal particles into cells is an important area of biological research in itself because it also relates to the cell’s communication with
Topical Review
a)
b)
c)
d)
Figure 3. Labelling of fixed cells with bioconjugated quantum dots. (a) Fixed human epithelial cells were first incubated with a mixture of biotinylated phalloidin and monoclonal anti-histone antibodies, which bind to the actin filaments and histone proteins in the cells, respectively. In a second step, commercially available green and red fluorescent polymer-coated quantum dots conjugated with streptavidin and protein A, which bind to the biotinylated phalloidin and the anti-histone antibodies, respectively, were added. In this way actin filaments and histone proteins can be visualized by green and red fluorescence. Image courtesy of Quantum Dot Corporation (http://www.qdots.com). (b) Detection of human epidermal growth factor receptor 2 (Her2) with commercially available red fluorescent polymer-coated quantum dots conjugated with streptavidin. Her2 receptors on the surface of living breast cancer cells (SK-BR-3) were labelled with streptavidin-decorated quantum dots after the cells were sequentially incubated with recombinant humanized anti-Her2 monoclonal antibodies and biotinylated goat anti-human IgG antibodies. Image courtesy of Quantum Dot Corporation. (c), (d) HEK cells with serotonin transporter proteins (hSERTs) expressed in their membrane have been incubated with green fluorescent mercaptoacetic-acid-coated CdSe/ZnS quantum dots conjugated with serotonin. (c) A differential interference contrast image, (d) with the identical field of view, a fluorescence image. Fluorescence labelling of the hSERTs in the membrane is clearly visible. Image courtesy of the group of Professor S Rosenthal. For experimental details see [93].
the outside world, to the uptake and processing of nutrients, and also to the uptake of viruses. Apart from that, cellular uptake mechanisms are important in a variety of applications including drug, nucleic acid and gene delivery as well as cell marking with iron oxide or gold nanoparticles. Therefore, cellular transport pathways have been studied extensively (the interested reader is referred to textbooks of cell biology or molecular biology; a recent review on cellular membrane traffic is [122]). Because cells need to communicate with the outside world and because they need to take up nutrients they are equipped with natural transport pathways both for incorporating and for secreting materials. In general, these transport pathways are part of a whole network of cellular membrane traffic, which generally involves membranesurrounded vesicles of aqueous content. Natural uptake into cells is called endocytosis (again, the reader is referred to textbooks of cell biology or molecular biology; recent reviews on endocytosis are [123, 124]). Depending on the size of endocytosed materials and on the detailed uptake mechanisms, various types of endocytosis are discriminated: pinocytosis for small particles and dissolved macromolecules (diameters up to about 150 nm), phagocytosis for larger structures such
as whole cells, cell debris or bacteria. Depending on the cellular molecules involved in endocytosis, clathrin-dependent and clathrin-independent pathways are discriminated. (In simple words, clathrin is a protein cage that assembles around cell membrane invaginations during endocytosis, forming the so-called clathrin-coated pits. The assembly unit of clathrin is a three-legged structure called triskelion, being a highly interesting tertiary structure of a protein.) Pinocytosis and endocytosis are often used as synonyms. One or the other mechanism can prevail depending on the cell type under consideration. For example, macrophages are cells specialized in clearing ‘large’ structures such as debris of dead cells or foreign intruders such as bacteria or colloidal nanoparticles. They do that by phagocytosis. While the mechanisms differ in details, their common feature is that cells ‘engulf’ the material to be incorporated. Invaginations of the cell membrane around the material finally lead to the budding of an intracellular membrane-surrounded vesicle. Dependent on the uptake mechanism and on the incorporated material, the fate of such vesicles differs. In pinocytosis for example these vesicles, which are called endosomes, mature or fuse to become lysosomes. Endosomes and lysosomes are responsible R17
Topical Review
for the breakdown of ingested materials. These vesicles are equipped with specialized enzymes that would degrade proteins and nucleic acids. Among the uptake mechanisms, fluid phase uptake and uptake of materials bound to the cell surface are discriminated. Fluid phase uptake is the continuous incorporation of solutes from the media cells live in. Uptake of bound material can be specific or unspecific in terms of the type of binding to cell surfaces. Receptormediated endocytosis is called specific because it involves and is triggered by a specific key/keyhole type receptor– ligand binding interaction. Among the many examples, a typical one is the uptake of transferrin via the transferrin receptor, which provides cells with their iron ‘diet’. In this case, receptor and ligand are recycled to the cell surface after intracellular release of iron. The transferrin/transferrin receptor system has been widely used in drug and gene delivery [125, 126]. Receptor-mediated endocytosis is also involved in cellular communication, called signalling. One example of medical relevance in tumour growth is the endocytosis of the urokinase-type plasminogen activator (uPA) receptor (uPA-R) upon binding of uPA. Without going into molecular details, this event gives the cell a signal to divide and/or to migrate [127]. Triggering uptake into cells upon binding does not necessarily require specific receptor–ligand type binding events. In most cases unspecific binding is sufficient and is easily accomplished with cationic materials or particles. These bind by simple electrostatic interaction to the outside of cells, which are generally negatively charged (negatively charged glycosaminoglycans of the extracellular matrix are responsible). However, it is well known that negatively charged particles are also taken up into cells, and in fact most cell labelling experiments with quantum dots have been carried out with anionic surface-coated particles. Aiming at exploiting cellular transport mechanisms such as endocytosis for cell labelling, it is highly instructive to consider the vast amount of literature available from the fields of drug and gene delivery. Just making cells fluorescent by making them ingest quantum dots is a simple task. Usually, these particles, as they cannot be degraded, will be stored in membrane-surrounded compartments that will not be accessible to solutes of the cytoplasm. Using quantum dots for labelling specific intracellular structures outside endocytosed vesicles or to even image cellular reactions in the cytoplasm or the nucleus will require more sophisticated tools. The same problem prevails in drug and particularly in nucleic acid/gene delivery [128]. To exert their desired effect, these macromolecules need to be transported to the cell nucleus in the majority of current applications (genes are transcribed into messenger RNA, mRNA, in the nucleus; mRNA then carries the sequence information into the cytoplasm which is there translated into protein sequences). For transport across cellular membranes nucleic acids are self-assembled (compacted) with polycations or cationic lipids to form charged nanoparticles (also called ‘complexes’) [128]. Such particles can bind to cell surfaces non-specifically via electrostatic interaction or specifically if equipped with receptor ligands. This can easily be accomplished by chemical coupling of ligand molecules to pre-assembled particles or to the cationic moiety before assembly of the particle [129]. Uptake into cells proceeds via endocytosis. Escape from internal vesicles can be mediated R18
by also incorporating fusogenic components into the complex such as membrane-disrupting peptides that unfold their activity during the internalization process [130–132]. Otherwise, the cationic moiety used to compact the polyanionic nucleic acid macromolecules can be chosen to comprise an inherent chemical structure that would interfere with the ‘standard’ degradation of internalized material. For example, the polycation polyethylenimine comprises secondary and tertiary amino groups that can buffer the natural acidification process within endosomes (the so-called ‘proton sponge effect’, [133]). This in turn leads to an osmotic destabilization of these vesicles, which together with the swelling of polyethylenimine at acidic pH leads to endosome disruption [134]. The chemical structures of selected lipids also promote membrane reorganizations that can be exploited to release internalized materials such as nucleic acids into the cytoplasm [135]. Exploiting cellular transport processes for ‘invasion’ has been copied from viruses, which evolved mechanisms of tricking cells for their own purposes to perfection. This excursion to nucleic acid and drug delivery and existing quantum dot literature leads us to conclude that natural transport processes can and should be exploited for quantum dot delivery to cells in an analogous manner. The involved surface chemistries are similar (see above); receptor ligands have been coupled to quantum dots and endocytotic uptake, receptor mediated [20, 35, 36, 38, 45, 101] as well as unspecific/adsorptive, has been described for these particles [23, 40, 42, 74, 91, 136]. The efficacy of uptake as well as the exact uptake mechanisms (receptor specific versus adsorptive/unspecific) certainly depend on a variety of parameters including particle size, surface chemistry, and, importantly, also the cell type under consideration. Experimental evidence for endocytotic uptake of quantum dots has been obtained by colocalization experiments with endosome markers. Hanaki et al used fluorescein-labelled dextrane as marker for endosomes/lysosomes [40] which were found to colocalize with ingested quantum dots. Jaiswal et al found colocalization of quantum dots with the endosome specific marker pECFP [42]. This group furthermore demonstrated that quantum dot uptake can be blocked by cooling the cells to 4 ◦ C, a technique known to block endocytosis. Coating the quantum dot surface with polyethylene glycol (PEG) reduces adsorption to the membrane and uptake of these particles by cells [45, 59]. Ligand-modified quantum dots that bind to membranebound receptors are internalized together with the receptor molecules [45, 101]. Lidke et al demonstrated that epidermalgrowth-factor- (EGF-) conjugated quantum dots bind to erbBX transmembrane receptors which are responsible for mediating cellular responses to EGF [101]. EGF-conjugated quantum dots bound to the erbBX receptors were internalized by the cells. The authors demonstrated this to be via clathrincoated pits by simultaneously adding fluorescence-labelled transferrin, which was found to colocalize with the quantum dots as well on the cell surface as after internalization in early endosomes. Most groups that studied cellular quantum dot uptake report that in cell cultures or animal models, at later stages quantum dots are stored in granular compartments around the nucleus [36, 40, 42, 70, 74], without penetrating the nucleus,
Topical Review
a)
b)
c)
Figure 4. (a) Uptake of colloidal quantum dots by living cells. MDA-MB-231 cells were grown on top of red fluorescent quantum dots embedded in a collagen layer. After about 4 h incubation the cells have ingested many quantum dots and stored them in intracellular vesicles close to the nucleus. The images show overlays of phase contrast images to visualize the cells and fluorescence images to visualize the accumulated quantum dots. The image was recorded by the authors together with R Boudreau and M Le Gros in the groups of Professors P Alivisatos and C Larabell. For experimental details see [74, 136]. (b) Tracing of cell lineage with colloidal quantum dots. Human breast carcinoma MDA-MB-435s and MCF-7 cells have been loaded with green and red fluorescent polymer-coated CdSe/ZnS quantum dots, respectively, prior to seeding both types of cells on the same substrate. The image shows an overlay of a phase contrast image to visualize the cells and a fluorescence image to visualize the incorporated quantum dots. By the colour of fluorescence the cells can be attributed to their type, i.e. cells with green fluorescence are known to be MDA-MB-435s cells. This image was recorded by Tim Liedl in the group of the authors. For experimental details see [74, 136]. (c) Phagokinetic tracks. MDA-MB-231 cells were grown on top of red fluorescent quantum dots embedded in a collagen layer. After about 1 day incubation trails free of quantum dots can be seen. These so-called phagokinetic tracks represent a blueprint of the migration pathway of the cells, along which all quantum dots have been ingested by the cells. The images show overlays of phase contrast images to visualize the cells and fluorescence images to visualize the accumulated quantum dots. The image was recorded by the authors together with R Boudreau and M Le Gros in the groups of Professors A P Alivisatos and C Larabell. For experimental details see [74, 136].
see figure 4(a). Hanaki et al observed that after cell division quantum dots can be found not only in the perinuclear region but also in the neighbourhood of the cell membrane [40]. Another general agreement is the fact that upon cell division ingested quantum dots are distributed between both daughter cells [40, 60, 70, 74]. This is again in analogy to what is observed in nucleic acid delivery. We conclude that great potential lies in further adopting the chemistries and technologies of advanced drug delivery for quantum dot delivery. This can lead to enhanced efficacy and specificity of labelling and may yield numerous novel applications. An excellent precedent is the latest developments in iron oxide nanoparticle delivery to cells [137, 138].
5. Tracking the path and the fate of individual cells by quantum dot labels Summarizing the above, quantum dots with suitable surface coatings can bind to cells non-specifically or specifically by molecular recognition, can be taken up into cells by natural transport processes, and can be stored within cells in membrane-surrounded structures. Hence, by virtue of their optical properties quantum dots can highlight cells and subcellular structures above the surrounding background; in other words, they add contrast to a material that otherwise provides little contrast under visible light observation; in yet
other words, they can communicate to the observer an image of a biological structure of interest. Synonymous with the term ‘label’, quantum dots can be classified as contrast agents, a term used in medical diagnostic imaging procedures for substances that help to distinguish an anatomical structure of interest from surrounding tissue or, in physical terms, to enhance the signal-to-noise ratio in a diagnostic image. This analogy is emphasized here, because diagnostic imaging with approved contrast agents also develops towards exploiting molecular recognition for selectively displaying ever-smaller structures and/or physiological functions. In this young science of molecular imaging similar requirements on the surface characteristics of contrast agents apply as discussed here for quantum dots as labels. On the other hand, the safety requirements imposed on contrast agents for medical imaging will apply for quantum dots. Without doubt, quantum dots will play a major role in molecular imaging if the issues related to their potential long term toxicities can be resolved. Some considerations will be presented at the end of this section. Recent literature demonstrates the great potential of quantum dots in molecular imaging. We will discuss two major areas of interest: the one is tracking the path and the fate of individual cells upon loading them with quantum dots, which is important for many biological, but also medical questions [139]. The other is administering quantum dots directly to the body in order to achieve accumulation in a R19
Topical Review
structure of interest such as a tumour or lymph node. This is in analogy to classical medical imaging techniques. One important example of the first area is embryonic development. This has been intensely studied in classic biology, then in molecular cell biology, and recently has again attracted much attention in stem cell research [140]. Important questions are how a complete organism can develop from one single cell or which cell in an early stage embryo will finally develop to which part of the final creature or which signals between cells during the early stages of development induce the specialization (‘differentiation’) of individual cells or groups of cells to ultimately become specialized tissues of an adult organism. Another important question is where cells would migrate (‘home’) and how they would differentiate and what their offspring cells would do if implanted into an adult organism. This is related to studying embryonic development, particularly if the administered cells are stem cells. Obviously, this is highly relevant in evaluating stem cell therapies. Another area where cell tracking plays an important role is cancer development and the spreading of cancer cells (metastasis). Cell labelling is a useful tool in these research areas. Until most recently, oil drops or organic fluorophores have been microinjected as markers [139] in embryonic development studies. Also fate-mapping studies have been carried out using fluorescence labelling of cells [141]. Another method is transfecting cells with reporter genes either coding for fluorescent proteins like GFP [142] or coding for some selectable functional marker [143]. More recently, the suitability of quantum dots for tracking and fate mapping has been demonstrated by several research groups. It needs to be emphasized again, that the outstanding optical properties of quantum dots such as reduced photobleaching and their long term stability (due to their non-degradability) are essential advantages of quantum dots over organic fluorophores in these long term observation studies. Most of these studies where concerned with tracking cancer cells. Without going much into biological details, Dubertret et al have shown the potential of quantum dot labels in embryonic development studies [60]. From a technological point of view, one has to discriminate between inside and outside labelling. As internal label, quantum dots can be either microinjected in cells [60] or ingested by cells [42, 70, 108]; as external label they can be attached to the cell membrane [38]. In a technology-oriented work and without going much into biological detail, Dubertret et al micro-injected quantum dot micelles in individual cells of Xenopus blastomeres and monitored the distribution of the label in the growing embryo. Several groups have used quantum dot labels that have been ingested by cells to follow the fate of these cells within cell cultures [42, 70, 108]. One example for such experiments is shown in figure 4(b). The experimental procedure is straightforward. First, cells of different lineage are incubated with quantum dots of different colour. The cells ingest the quantum dots, and after a certain incubation time non-ingested quantum dots are removed by exchanging the culture medium. The labelled cells are then seeded as a co-culture on a new substrate. In this way the lineage of each cell can be determined by its colour of fluorescence by optical microscopy. Such noninvasive fluorescence labelling is maybe the easiest way to distinguish cell lines in co-cultures, since most often the lineage of cells R20
cannot be simply determined by the morphology of the cell. The method is easy to use and a kit for labelling of different cell lines with different colours of fluorescence is commercially available [70]. By varying the ratio of the different quantum dot colours with which cells are labelled, many different codes can be obtained, and in this way many different cell lines can be tagged with a unique label [70]. Quantum dot labelling has been used for example to label five different subpopulations of Chinese hamster ovary carcinoma (CHO) cells on one substrate [70], to follow the fate of three subpopulations of Dictyosteluim discoideum cells in one cell colony [42], and to monitor the migration of primary hepatocyte cells in a coculture with 3T3 fibroblasts [108]. Another important area of research is cell adhesion and cell migration. These play essential roles in cancer development [144] and in designing biocompatible surfaces of medical implants relevant in orthopaedics and in tissue engineering for example. Quantum dots can be used to study cell migration in an indirect way. As early as 1976 Albrecht– Buehler introduced a method called phagokinetic track to follow cellular migration on cell culture substrates [145– 147]. The idea is based on the use of a surface coated with markers, on which cells are seeded. Upon migration along the surface cells internalize and remove the marker, and marker-free zones are practically a blueprint of their pathway. Originally gold colloids were used as markers [147], and they were visualized by dark-field microscopy or by transmission electron microscopy. This method has been improved over time and has been used for a whole variety of mobility/migration studies [148–150]. Besides colloidal gold polystyrene micro-beads [151] have also been used as marker. Also quantum dots have been used successfully for recording phagokinetic tracks [74, 136]. Due to their small size, improved spatial resolution can be expected. Furthermore, tracks could be observed in stacked layers with different colours of fluorescence, which would allow for analysing migration behaviour in three-dimensional culture. One example of phagokinetic tracks is shown in figure 4(c).
6. Using quantum dots as contrast agents The second area of major interest mentioned above was administering quantum dots as contrast agents directly to the body for imaging purposes. Such images are generated by exposing a body or part of a body to electromagnetic radiation or ultrasound and by measuring a resulting signal generated in the body, in many instances with the help of contrast agents. Typical examples are magnetic resonance imaging (MRI), x-ray scans, radioactive imaging techniques such as scintigraphy and positron emission tomography (PET) or ultrasound diagnostics. Typical contrast agents are paramagnetic iron oxide nanoparticles for MRI, halogenated organic compounds for x-ray, fluoro-deoxyglucose for PET or fluorocarbon-filled micro-bubbles for ultrasound diagnostics. A number of highly promising studies have been published that combine elegant synthetic chemistries to adapt the known procedures of drug targeting with the advantageous physical characteristics of quantum dots and with advanced detection technologies. It is instructive to briefly review what is required to accumulate a substance in a target structure or target organ
Topical Review
in a living animal or a human. It is obvious that such preferential accumulation is important if one wants to produce an image of a target structure with the help of a contrast agent. Targeted accumulation is also important because any ingested or administered substance will reach a level of toxicity at some dose. A great proportion of drugs in medical application are administered systemically; that is, they distribute throughout the body and potentially act throughout the body and therefore have, among other reasons, undesired side effects [152]. For accumulating a substance in a target structure, there are several possibilities. One is direct injection into a target organ where the substance may diffuse over a limited distance and may otherwise be retained there. Another option is passive targeting, that is administering a substance systemically but exploiting its biophysical properties that will lead to passive accumulation in a target organ. The third is active targeting, that is again administering a substance systemically, again exploiting its biophysical properties but in addition using molecular recognition (receptor–ligand type interactions) to accumulate the substance at the target site. Once at the target site, the substance may enter cells by natural transport mechanisms as discussed above or may remain extracellular, interacting with the extracellular matrix. For active and passive targeting, a general prerequisite is sufficiently long circulation time in the blood stream. Otherwise the substance will be cleared from the circulation by the body’s defence systems before it has a chance to accumulate at a target site and/or to find its cognate receptor (the reticulo-endothelial system, RES, is specialized in clearing foreign intruders). The analogy between quantum dot and drug delivery has already been mentioned. In both disciplines, long blood circulation times can be achieved by suitable surface characteristics which can be obtained by surface coatings with polyethyleneglycol of a certain minimal chain length, for example [59]. Such coatings enhance colloidal stability and reduce undesired interactions with blood components [153]. Active targeting can be achieved by incorporating receptor ligands into the surface coating. These principles have been exploited for using quantum dots as contrast agents in imaging with optical detection. One general problem for optical detection is the light absorption in the tissue. Larson et al have injected fluorescent CdSe/ZnS quantum dots into the tail veins of mice [84]. The particle distribution within the animal was recorded with multiphoton microscopy. Multiphoton excitation enables imaging with thick specimens. Quantum dots have a high two-photon absorption cross section and also suffer less from photobleaching compared to standard organic fluorophores, which makes them good labels for multiphoton microscopy. With this technique the authors could image the capillary structure of blood vessels through the skin and measure the velocity of the blood flow. In another study Ballou et al injected CdSe/ZnS quantum dots which were fluorescent in the visible into the tail veins of nude mice [59]. Again, the particles could be visualized by fluorescence imaging. Particles in the animals remained fluorescent at least for four months. For a certain time, the particles circulated in the blood stream until they were deposited in liver (RES probably), skin, bone marrow, spleen, and lymph nodes. Finally the deposition in the lymph nodes and the bone marrow was enhanced relative to the deposition in
the liver. The authors claim that quantum dots were also seen in the intestinal contents due to excretion from the liver. It is however unclear how this conclusion is derived from the shown data (a fluorescent intestine does not necessarily mean that the intestinal content is fluorescent. And how would excretion of a substance from the liver into the intestinal content work?). The important lesson from this paper is probably that the rate and location of the deposition are dependent on the surface coating of the quantum dots. Long blood circulation times were achieved with surface coatings with polyethyleneglycol of a certain minimal chain length. This highlights again the already discussed analogy to drug and gene delivery, where qualitatively the same observations have been made. Recently, quantum dots fluorescent in the infrared have also been employed as contrast agent for optical detection. As a contrast label, quantum dots with fluorescence in the infrared are of particular importance, because their emission can be chosen in a spectral window in which absorption by the tissue is minimum [90, 154]. One exciting medical application of these particles has been demonstrated recently by Kim et al [155] who showed what can be achieved with local administration of quantum dots as contrast agents. The authors injected quantum dots intradermally in the paw of a mouse and in the thigh of a pig. The particles were then rapidly taken up by lymphatic collecting vessels passing to the sentinel nodes in the axilla of the mouse and in the groin of the pig. This combination of local administration followed by passive targeting could be used for intra-operative lymph node mapping using an imaging system also developed by the authors. This is important in various clinical settings, for example in surgical removal of tissue afflicted with tumour cells. The quantum dots as contrast agents can be used for sentinel lymph node biopsy [18]. In this method node regional fields are staged for metastasis. Lymphatic drainage is mapped from a primary tumour site to the draining lymph node or nodes, and these are then removed. If the sentinel node is normal, all of the lymph nodes in the node field can be assumed to be normal. Compared to the currently used contrast agent for this operation, isosulfan blue, infrared fluorescent quantum dots offer several advantages. First, their fluorescence can be recorded through the skin, which gives the surgeon guidance even before making the incision. The surgeon can identify the position of the quantum dots and thus the lymph channels in real time and in this way the size of the incision can be reduced. Also the complete removal of the lymph node can be controlled easily by testing the remaining tissue for further fluorescence. This method seems to have a very promising perspective, although also here toxicity issues still have to be clarified. In their most recent paper Gao et al from the Nie group describe multifunctional quantum dot probes for cancer targeting and imaging in living animals [156]. They also have integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging, which allows high sensitivity detection of molecular targets in vivo. These researchers have generated the required colloidal stability of their quantum dots with a sophisticated surface coating, which did not interfere with the optical properties of their probes. They exploited passive targeting to experimental tumours upon intravenous administration by virtue of the leaky nature of tumour blood vessels and in addition performed R21
Topical Review
active targeting by coupling a tumour-specific antibody to the quantum dot probes. They achieved excellent accumulation of these probes in the tumours, which made high sensitivity specific imaging of this ‘structure’ possible. The reported results and procedures also contain highly interesting side aspects. These authors also used their probes to directly label specific cells in culture before these cells were inoculated in the animals in order to grow tumours. They observed that their probes accumulated in the cell nuclei of the treated cells within a short period of time, a feature not observed with different probes by other groups (see above) which may be useful for sensing and imaging biological processes in this cellular compartment. Inoculated in animals, the specific cells could be tracked by virtue of their fluorescence. This is an elegant combination of cell tracking and imaging with systemically applied quantum dot contrast agents and all this with the same particle type in one experiment. This paper is also important because the authors provide quantitative information on which dose of quantum dot probe is required to obtain a particular signal. Summarizing the above, there is no doubt about the great potential of quantum dots as labels or contrast agents in life sciences and medical diagnostics. Future studies will have to characterize the dose–response relationships of quantum dots. Dose–response in this case means not only evaluating which minimal dose is needed to obtain the desired optical signal as a response but also which dose is acceptable in terms of undesired side effects, which are a type of response as well. The existing literature gives some hints that at least in short-term studies toxicity may not be a limiting problem. Medical application in humans will however require an unequivocal assessment of the potential long term effects of administered quantum dots. Together with other interdisciplinary developments of modern science, the optimization and application of quantum dots in biology and medicine may revolutionize what we know and what we think about biological processes.
7. Conclusion Five years after the first labelling experiments of cells with colloidal quantum dots by the groups of Alivisatos [52] and Nie [35] a new generation of experiments with quantum dots has just started. Whereas the first studies have to be regarded rather as demonstration experiments, in which the capability of a new technology was reported, nowadays colloidal quantum dots are used to address specific biological questions. Besides high quality labelling of cellular compartments [56] time-resolved single-molecule studies are of special importance [78, 101]. Routine medical diagnostic procedures usually image macroscopic structures. Imaging microscopic structures, physiological functions and molecular processes is a major aim of the young science of molecular imaging, which of course is not limited to future medical applications but plays an important role in basic life sciences. Recent literature demonstrates the great potential of quantum dots for ‘imaging’ purposes [155, 156]. If the issues related to the potential toxicity of quantum can be resolved, these materials will also become valuable tools for medical imaging. R22
Acknowledgments The authors are grateful to Christian Albrecht, Tim Liedl, Stefan Kudera, Bernd Zebli, Christian Kirchner, and Dr Valentina Emiliani for critically reading the manuscript and helpful discussions and to Professors P Alivisatos, C Larabell, S Rosenthal, H Gaub and Dr Bruchez for helpful discussions. This work was funded by the Emmy Noether program of the German research foundation DFG (WJP), the Fonds der Chemischen Industrie (WJP), the Marie Curie training program of the European Union (TP), and the University of Bari (TP).
References [1] Stephens D J and Allan V J 2003 Light microscopy techniques for live cell imaging Science 300 82–6 [2] Weijer C J 2003 Visualizing signals moving in cells Science 300 96–100 [3] Miyawaki A, Sawano A and Kogure T 2003 Lighting up cells: labelling proteins with fluorophores Nat. Cell Biol. 5 S1–7 [4] Geiger B and Volberg T 1994 Conjugation of fluorescent dyes to antibodies Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 387–93 [5] Kulzer F et al 1997 Single-molecule optical switching of terrylene in p-terphenyl Nature 387 688–91 [6] Dickson R M et al 1997 On/off blinking and switcing behaviour of single molecules of green fluorescent protein Nature 388 355–8 [7] Pawley J B and Centonze V E 1994 Practical laser-scanning confocal light microscopy: obtaining optimal performance from your instrument Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 44–64 [8] Hermann R, Walther P and M¨uller M 1996 Immunogold labeling in scanning electron microscopy Histochem. Cell Biol. 106 31–9 [9] Roth J 1996 The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry Histochem. Cell Biol. 106 1–8 [10] Koster A J and Klumperman J 2003 Electron microscopy in cell biology: integrating structure and function Nat. Cell Biol. 5 SS6–10 [11] Cognet L et al 2003 Single metallic nanoparticle imaging for protein detection in cells Proc. Natl Acad. Sci. USA 100 11350–5 [12] Mitchell P 2001 Turning the spotlight on cellular imaging Nat. Biotechnol. 19 1013–7 [13] Klarreich E 2001 Biologists join the dots Nature 413 450–2 [14] Jovin T M 2003 Quantum dots finally come of age Nat. Biotechnol. 21 32–3 [15] Seydel C 2003 Quantum dots get wet Science 3000 80–1 [16] Taton T A 2003 Bio-nanotechnology: two-way traffic Nat. Mater. 2 73–4 [17] Bentolila L A and Weiss S 2003 Biological quantum dots go live Phys. World 16 (3) 23–4 [18] Uren R F 2004 Cancer surgery joins the dots Nat. Biotechnol. 22 38–9 [19] Michalet X et al 2001 Properties of fluorescent semiconductor nanocrystals and their application to biological labeling Single Mol. 2 261–76 [20] Chan W C W et al 2002 Luminescent quantum dots for multiplexed biological detection and imaging Curr. Opin. Biotechnol. 13 40–6 [21] Sutherland A J 2002 Quantum dots as luminescent probes in biological systems Curr. Opin. Solid State Mater. Sci. 6 365–70 [22] Watson A, Wu X and Bruchez M 2003 Lighting up cells with quantum dots, biotechniques Biotechniques 34 296–303
Topical Review
[23] Parak W J et al 2003 Biological applications of colloidal nanocrystals Nanotechnology 14 R15–27 [24] Gao X and Nie S 2003 Molecular profiling of single cells and tissue specimens with quantum dots Trends Biotechnol. 21 371–4 [25] Bagwe R P, Zhao X and Tan W 2003 Bioconjugated luminescent nanoparticles for biological applications J. Dispersion Sci. Technol. 24 453–64 [26] Dubertret B 2003 In vivo imaging using quantum dots J. Med. Sci. 19 532–4 [27] Alivisatos P 2004 The use of nanocrystals in biological detection Nat. Biotechnol. 22 47–51 [28] Pellegrino T et al 2005 On the development of colloidal nanoparticles towards multifunctional structures and their possible use for biological applications Small 1 48–63 [29] Dabbousi B O et al 1997 (CdSe)ZnS core-shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites J. Phys. Chem. B 101 9463–75 [30] Talapin D V et al 2001 Highly luminescent monodisperse CdSe and CdSe/ZnS nanocrystals synthesized in a hexadecylamine–trioctylphosphine oxide–trioctylphospine mixture Nano Lett. 1 207–11 [31] Peng Z A and Peng X 2001 Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor J. Am. Chem. Soc. 123 183–4 [32] Reiss P, Bleuse J and Pron A 2002 Highly luminescent CdSe/ZnSe core/shell nanocrystals of low size dispersion Nano Lett. 2 781–4 [33] Parak W J et al 2002 Conjugation of DNA to silanized colloidal semiconductor nanocrystaline quantum dots Chem. Mater. 14 2113–9 [34] Wilhelm C et al 2003 Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating Biomaterials 24 1001–11 [35] Chan W C W and Nie S 1998 Quantum dot bioconjugates for ultrasensitive nonisotopic detection Science 281 2016–8 [36] Zhang C Y et al 2000 Quantum dot-labeled trichosanthin Analyst 125 1029–31 [37] Winter J O et al 2001 Recognition molecule directed interfacing between semiconductor quantum dots and nerve cells Adv. Mater. 13 1673–7 [38] Kloepfer J A et al 2003 Quantum dots as strain- and metabolism-specific microbiological lables Appl. Environ. Microbiol. 69 4205–13 [39] Mitchell G P, Mirkin C A and Letsinger R L 1999 Programmed assembly of DNA functionalized quantum dots J. Am. Chem. Soc. 121 8122–3 [40] Hanaki K et al 2003 Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker Biochem. Biophys. Res. Commun. 302 496–501 [41] Chen C-C et al 1999 Self-assembly of monolayers of cadmium selenide nanocrystals with dual color emission Langmuir 15 6845–50 [42] Jaiswal J K et al 2003 Long-term multiple color imaging of live cells using quantum dot bioconjugates Nat. Biotechnol. 21 47–51 [43] Sukhanova A, Venteo L, Devy J, Artemyev M, Oleinikov V, Pluot M and Nabiev I 2002 Highly stable fluorescent nanocrystals as a novel class of labels for immunohistochemical analysis of paraffin-embedded tissue sections Lab. Investigation 82 1259–61 [44] Sukhanova A et al 2004 Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells Anal. Biochem. 324 60–7 [45] Pinaud F et al 2004 Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides J. Am. Chem. Soc. 126 6115–23 [46] Pathak S et al 2001 Hydroxylated quantum dots as luminescent probes for in situ hybridization J. Am. Chem. Soc. 123 4103–4
[47] Wang Y A et al 2002 Stabilization of inorganic nanocrystals by organic dendrons J. Am. Chem. Soc. 124 2293–8 [48] Skaff H and Emrick T 2003 The use of 4-substituted pyridines to afford amphiphilic, pegylated cadmium selenide nanoparticles Chem. Commun. 2003 52–3 [49] Aldana J, Wang Y A and Peng X 2001 Photochemical instability of CdSe nanocrystals coated by hydrophilic thiols J. Am. Chem. Soc. 123 8844–50 [50] Gerion D et al 2001 Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ZnS semiconductor quantum dots J. Phys. Chem. B 105 8861–71 [51] Giersig M et al 1997 Chemistry of nanosized silica-coated metal particles-EM-study Ber. Bunsenges. Phys. Chem. 101 1617–20 [52] Bruchez M J et al 1998 Semiconductor nanocrystals as fluorescent biological labels Science 281 2013–6 [53] Correa-Duarte M A, Giersig M and Liz-Marz´an L M 1998 Stabilization of CdS semiconductor nanoparticles against photodegradation by a silica coating procedure Chem. Phys. Lett. 286 497–501 [54] Mulvaney P et al 2000 Silica encapsulation of quantum dots and metal clusters J. Mater. Chem. 10 1259–70 [55] Schroedter A et al 2002 Biofunctionalization of silica-coated CdTe and gold nanocrystals Nano Lett. 2 1363–7 [56] Wu M X et al 2003 Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots Nat. Biotechnol. 21 41–6 [57] Pellegrino T et al 2004 Hydrophobic nanocrystals coated with an amphiphilic polymer shell: a general route to water soluble nanocrystals Nano Lett. 4 703–7 [58] Petruska M A, Bartko A P and Klimov V I 2004 An amphiphilic approach to nanocrystal quantum dot-titania nanocomposites J. Am. Chem. Soc. 126 714–5 [59] Ballou B et al 2004 Noninvasive imaging of quantum dots in mice Bioconjugate Chem. 15 79–86 [60] Dubertret B et al 2002 In vivo imaging of quantum dots encapsulated in phospholipid micelles Science 298 1759–62 [61] Parak W J et al 2004 Quantum dots Nanoparticles—From Theory to Application ed G Schmid (Weinheim: Wiley–VCH) pp 4–49 [62] Bawendi M G, Steigerwald M L and Brus L E 1990 The quantum mechanics of large semiconductor clusters (‘quantum dots’) Annu. Rev. Phys. Chem. 41 477–96 [63] Alivisatos A P 1996 Semiconductor clusters, nanocrystals, and quantum dots Science 271 933–7 [64] Alivisatos A P 1997 Nanocrystals: building blocks for modern materials design Endeavour 21 56–60 [65] Efros A L and Rosen M 2000 The electronic structure of semiconductor nanocrystals Annu. Rev. Mater. Sci. 30 475–521 [66] Murray C B, Kagan C R and Bawendi M G 2000 Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies Annu. Rev. Mater. Sci. 30 545–610 [67] Qu L and Peng X 2002 Control of photoluminescence properties of CdSe nanocrystals in growth J. Am. Chem. Soc. 124 2049–55 [68] Kippeny T, Swafford L A and Rosenthal S J 2002 Semiconductor nanocrystals: a powerful visual aid for introducing the particle in a box J. Chem. Educ. 79 1094 [69] Yu W W et al 2003 Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals Chem. Mater. 15 2854–60 [70] Mattheakis L C et al 2004 Optical coding of mammalian cells using semiconductor quantum dots Anal. Biochem. 327 200–8 [71] Rosenthal S J 2001 Bar-coding biomolecules with fluorescent nanocrystals Nat. Biotechnol. 19 621–2 [72] Han M et al 2001 Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules Nat. Biotechnol. 19 631–5
R23
Topical Review
[73] Ness J M et al 2003 Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection J. Histochem. Cytochem. 51 981–7 [74] Parak W J et al 2002 Cell motility and metastatic potential studies based on quantum dot imaging of phagokinetic tracks Adv. Mater. 14 882–5 [75] Tokumasu F and Dvorak J 2003 Development and application of quantum dots for immunocytochemistry of human erythrocytes J. Microsc. 211 256–61 [76] Cordero S R et al 2000 Photo-activated luminescence of CdSe quantum dot monolayers J. Phys. Chem. B 104 12137–42 [77] Jones M et al 2003 Photoenhancement of luminescence in colloidal CdSe quantum dot solutions J. Phys. Chem. B 107 11346–52 [78] Dahan M et al 2003 Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking Science 302 442–5 [79] Nirmal M et al 1996 Fluorescence intermittency in single cadmium selenide nanocrystals Nature 383 802–4 [80] Efros A L and Rosen M 1997 Random telegraph signal in the photoluminescence intensity of a single quantum dot Phys. Rev. Lett. 78 1110–3 [81] Kuno M et al 2000 Nonexponential ‘blinking’ kinetics of single CdSe quantum dots: a universal power law behavior J. Chem. Phys. 112 3117–20 [82] Koberling F, Mews A and Basch´e T 2001 Oxygen-induced blinking of single CdSe nanocrystals Adv. Mater. 13 672–6 [83] van Sark W G J H M et al 2002 Blueing, bleaching, and blinking of single CdSe/ZnS quantum dots Chem. Phys. Chem. 3 871–9 [84] Larson D R et al 2003 Water-soluble quantum dots for multiphoton fluorescence imaging in vivo Science 300 1434–6 [85] Hu J et al 2001 Linearly polarized emission from colloidal semiconductor quantum rods Science 292 2060–3 [86] Willard D M and Orden A V 2003 Resonant energy-transfer sensor Nat. Mater. 2 575–6 [87] Willard D M et al 2001 CdSe–ZnS quantum dots as resonance energy transfer donors in a model protein–protein binding assay Nano Lett. 1 469–74 [88] Wang S et al 2002 Antigen/antibody immunocomplex from CdTe nanoparticle bioconjugates Nano Lett. 2 817–22 [89] Medintz I L et al 2003 Self-assembled nanoscale biosensors based on quantum dot FRET donors Nat. Mater. 2 630–8 [90] Lim Y T et al 2003 Selection of quantum dot wavelengths for biomedical assays and imaging Mol. Imaging 2 50–64 [91] Dahan M et al 2001 Time-gated biological imaging by use of colloidal quantum dots Opt. Lett. 26 825–7 [92] Akerman M E et al 2002 Nanocrystal targeting in vivo Proc. Natl Acad. Sci. USA 99 12617–21 [93] Rosenthal S J et al 2002 Targeting cell surface receptors with ligand-conjugated nanocrystals J. Am. Chem. Soc. 124 4586–94 [94] Mahtab R, Harden H H and Murphy C J 2000 Temperature- and salt-dependent binding of long DNA to protein-sized quantum dots: thermodynamics of ‘inorganic protein’–DNA interactions J. Am. Chem. Soc. 122 14–7 [95] Mattoussi H et al 2001 Bioconjugation of highly luminescent colloidal CdSe–ZnS quantum dots with an engineered two-domain recombinant protein Phys. Status Solidi b 224 277–83 [96] Goldman E R et al 2002 Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays Anal. Chem. 74 841–7 [97] Goldman E R et al 2002 Avidin: a natural bridge for quantum dot-antibody conjugates J. Am. Chem. Soc. 124 6378–82 [98] Hermanson G T 1996 Bioconjugate Techniques (San Diego, CA: Academic) [99] Gerion D et al 2002 Sorting fluorescent nanocrystals with DNA J. Am. Chem. Soc. 124 7070–4
R24
[100] Gerion D et al 2003 Ultra-fast room-temperature single nucleotide polymorphism detection and multi-allele DNA detection using fluorescent nanocrystal probes and microarray Anal. Chem. 75 4766–72 [101] Lidke D S et al 2004 Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction Nat. Biotechnol. 22 198–203 [102] Demers L M et al 2000 A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles Anal. Chem. 72 5535–41 [103] Kondoh M et al 2002 Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells Toxicology 170 111–7 [104] Shen H-M, Yang C-F and Ong C-N 1999 Sodium selenite-induced oxidative stress and apoptosis in human hepatoma HepG2 cells Int. J. Cancer 81 820–8 [105] Dameron C T et al 1989 Biosyntesis of cadmium sulphide quantum semiconductor crystallites Nature 338 596–7 [106] Williams P, Keshavarz-Moore E and Dunnill P 2002 Schizosaccharomyces pombe fed-batch culture in the presence of cadmium for the production of cadmium sulphide quantum semiconductor dots Enzyme Microbiol. Technol. 30 354–62 [107] Kowshik M et al 2002 Microbial synthesis of semiconductor PbS nanocrystallites Adv. Mater. 14 815–8 [108] Derfus A M, Chan W C W and Bhatia S N 2004 Probing the cytotoxicity of semiconductor quantum dots Nano Lett. 4 11–8 [109] Santone K S, Acosta D and Bruckner J V 1982 J. Toxicol. Environ. Health 10 169–77 [110] Rikans L E and Yamano T 2000 Mechanisms of cadmium-mediated acute hepatotoxicity J. Biochem. Mol. Toxicol. 14 110–7 [111] Schulze E et al 1995 Cellular uptake and trafficking of a prototypical magnetic iron oxide label in vitro Investigative Radiol. 30 604–10 [112] Weissleder R et al 1989 Superparamagnetic iron oxide: pharmacokinetics and toxicity AJR Am. J. Roentgenol. 152 167–73 [113] Whitaker M 1994 Fluorescence imaging in living cells Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 37–43 [114] Wang Y-L 1994 Microinjection of proteins into somatic cells: needle microinjection and scrape loading Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 16–21 [115] Herr S et al 1994 Electropration of cells Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 37–43 [116] Chakrabarti R and Schuster S M 1994 Electroporation of antibodies into mammalian cells Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 44–9 [117] Osborn M 1994 Immunofluorescence microscopy of cultured cells Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 347–54 [118] Herzog M et al 1994 Immunofluorescence microscopy of the cytoskeleton: double and triple immonofluorescence Cell Biology—A Laboratory Handbook ed J E Celis (San Diego, CA: Academic) pp 355–60 [119] Wu M X et al 2003 Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots—corrigenda Nat. Biotechnol. 21 452 [120] Dumoulin A et al 2000 Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons Eur. J. Neurosci. 12 3883–92 [121] Meier J et al 2001 Fast and reversible trapping of surface glycine receptors by gephyrin Nat. Neurosci. 4 253–60 [122] Munro S 2004 Organelle identity and the organization of membrane traffic Nat. Cell Biol. 6 469–72 [123] Mousavi S A et al 2004 Clathrin-dependent endocytosis Biochem. J. 377 1–16
Topical Review
[124] Pelkmans L and Helenius A 2003 Insider information: what viruses tell us about endocytosis Curr. Opin. Cell Biol. 15 414–22 [125] Wagner E, Curiel D and Cotten M 1994 Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis Adv. Drug Deliv. Rev. 14 113–35 [126] Widera A, Norouziyan F and Shen W C 2003 Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery Adv. Drug Deliv. Rev. 55 1439–66 [127] Reuning U et al 2003 Urokinase-type plasminogen activator (uPA) and its receptor (uPAR): development of antagonists of uPA/uPAR interaction and their effects in vitro and in vivo Curr. Pharm. Des. 9 1529–43 [128] Niidome T and Huang L 2002 Gene therapy progress and prospects: nonviral vectors Gene Ther. 9 1647–52 [129] Cotten M and Wagner E 1993 Non-viral approaches to gene therapy Curr. Opin. Biotechnol. 4 705–10 [130] Wagner E et al 1992 Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle Proc. Natl Acad. Sci. USA 89 7934–8 [131] Plank C et al 1994 The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems J. Biol. Chem. 269 12918–24 [132] Plank C, Zauner W and Wagner E 1998 Application of membrane-active peptides for drug and gene delivery across cellular membranes Adv. Drug Deliv. Rev. 34 21–35 [133] Boussif O et al 1995 A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine Proc. Natl Acad. Sci. USA 92 7297–301 [134] Sonawane N D, Szoka F C Jr and Verkman A S 2003 Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes J. Biol. Chem. 278 44826–31 [135] Xu Y and Szoka F C Jr 1996 Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection Biochemistry 35 5616–23 [136] Pellegrino T et al 2003 Quantum dot-based cell motility assay Differentiation 71 542–8 [137] Lewin M et al 2000 Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells Nat. Biotechnol. 18 410–4 [138] Weissleder R et al 2000 In vivo magnetic resonance imaging of transgene expression Nat. Med. 6 351–5 [139] Pearson H 2002 Developmental biology: your destiny, from day one Nature 417 14–5 [140] Kubo A et al 2004 Development of definitive endoderm from embryonic stem cells in culture Development 131 1651–62
[141] Ferrari A et al 2001 In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye J. Biomed. Mater. Res. 56 361–7 [142] Tombolini R and Jansson J K 1998 Monitoring of GFP-tagged bacterial cells Bioluminescence Methods and Protocols ed R A LaRossa (Totowa, NJ: Humana Press) pp 285–98 [143] Brenner M K et al 1993 Gene-marking to trace origin of relapse after autologous bone-marrow transplantation Lancet 341 85–6 [144] Fuchs M et al 2004 Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein Cell Motil. Cytoskeleton 59 50–61 [145] Albrecht-Buehler G 1977 The phagokinetic tracks of 3T3 cells Cell 11 395–404 [146] Albrecht-Buehler G 1977 Phagokinetic tracks of 3T3 cells: parallels between the orientation of track segments and of cellular structures which contain actin or tubulin Cell 12 333–9 [147] Albrecht-Buehler G and Lancaster R M 1976 A quantitative description of the extenion and retraction of surface protrusions in spreading 3T3 mouse fibroblasts J. Cell Biol. 71 370–82 [148] Chen J D et al 1994 Human keratincytes make uniquely linear phagokinetic tracks Dermatology 188 6–12 [149] Stokes C L, Lauffenburger D A and Williams S K 1991 Migration of individual microvessel endothelial cells: stochastic model and parameter measurement J. Cell Sci. 99 41–430 [150] Scott W N, McCool K and Nelson J 2000 Improved method for the production of gold colloid monolayers for use in the phagokinetic track assay for cell motility Anal. Biochem. 287 343–4 [151] Obeso J L and Auerbach R 1984 A new microtechnique for quantitating cell movement in vitro using polystyrene bead monolayers J. Immunol. Methods 70 141–52 [152] Plank C et al 2003 Enhancing and targeting nucleic acid delivery by magnetic force Expert Opin. Biol. Ther. 3 745–58 [153] Finsinger D et al 2000 Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery Gene Ther. 7 1183–92 [154] Kim S et al 2003 Type-II quantum dots: CdTe/CdSe(Core/Shell) and CdSe/ZnTe(Core/Shell) heterostructures J. Am. Chem. Soc. 125 11466–7 [155] Kim S et al 2004 Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping Nat. Biotechnol. 22 93–7 [156] Gao X et al 2004 In vivo cancer targeting and imaging with semiconductor quantum dots Nat. Biotechnol. 22 969–76
R25
