Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging

To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using (125)I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1-2 d; in vivo imaging will take another 1-2 d depending on the experimental design.     

From analog to digital: exploring cell dynamics with single quantum dots

Semiconductor quantum dots (QDs) have emerged as new fluorescent probes for biology. When combined with ultrasensitive optical techniques, they allow motions of individual biomolecules to be tracked in live cells with high signal-to-noise and over unprecedented durations. Single QD imaging readily offers a powerful tool to investigate the organization in cell membranes. Altogether QDs will contribute to more advanced biological imaging and enable new studies on the dynamics of cellular processes.Robert Feulgen Lecture 2005 presented at the Joint Meeting of the Society for Histochemistry and The Histochemical Society in Noordwijkerhout, The Netherlands     

The effects of ABA on channel-mediated K(+) transport across higher plant roots

The transport and accumulation of K(+) in higher plant roots is regulated by ABA. Molecular and electrophysiological techniques have identified a number of discrete transporters which are involved in the translocation of K(+) from the soil solution to the shoots of higher plants. Furthermore, recent reports have shown that ABA regulates K(+) channel activity in maize and Arabidopsis roots which suggests that ABA regulation of K(+) transport in roots is, at least in part, ion channel-mediated. The signalling processes which underlie the ABA regulation of K(+) channels have been investigated. The effects of ABA on the membrane potential of intact maize root cells were also studied. It was found that ABA regulated the membrane potential of root cells and that this regulation is consistent with the hypothesis that ABA-induced K(+) accumulation in roots is mediated by K(+) channels.     

The different effects of PEG 6000 and NaCI on leaf development are associated with differential inhibition of root water transport

The inhibitory effects of PEG on whole-plant growth can exceed the effects of other osmolytes such as NaCI, and this has been ascribed to toxic contaminants, or to reduced oxygen availability in PEG solutions. We investigated another possibility, namely that PEG has an additional inhibitory effect on root water transport which in turn affects leaf development. The effects on first-leaf growth of applications of PEG 6000 or isoosmotic NaCI to the roots were determined using hydroponically grown maize (Zea mays L.) seedlings. Leaf growth rates were inhibited within minutes of PEG application to the roots and remained inhibited for days. The inhibitory effects on growth of NaCI, and also of KCl and mannitol, were much smaller. The comparative effects of NaCI and PEG on root water transport were determined by assaying pressurized flow through excised roots. PEG induced a 7-fold greater inhibition of flow through live roots than NaCI. Killing of the roots by heat treatment, to reduce cell membrane resistances to solute penetration, nearly doubled the flow rate for roots in NaCI, but not for roots in PEG. We suggest that the greater viscosity of PEG solutions, as compared with NaCI, may be a primary factor contributing to the additional inhibition of water flow through live and killed roots. PEG did not have additional effects on leaf turgor but had a 3 times greater inhibitory effect than NaCI on the irreversible extensibility of the leaves and induced 16 times more leaf accumulation of the growth inhibitory stress hormone abscisic acid (ABA). We conclude that greater inhibition of root water transport by PEG 6000, as compared with NaCI, leads to additional reductions in extensibility, additional ABA accumulation, and a greater inhibition of leaf growth.     

Imaging Depths of Near-infrared Quantum Dots in First and Second Optical Windows

AbstractPotential advantages of quantum dot (QD) imaging in the second optical window (SOW) at 1,000 to 1,400 nm over the first optical window (FOW) at 700 to 900 nm have attracted much interest. QDs that emit at 800 nm (800QDs) and QDs that emit at 1,300 nm (1,300QDs) are used to investigate the imaging depths at the FOW and SOW. QD images in biologic tissues are processed binarized via global thresholding method, and the imaging depths are determined using the criteria of contrast to noise ratio and relative apparent size. Owing to the reduced scattering in the SOW, imaging depth in skin can be extended by approximately three times for 1,300QD/SOW over 800QD/FOW. In liver, excitation of 1,300QD/SOW can be shifted to longer wavelengths; thus, the imaging depth can be extended by 1.4 times. Effects of quantum yield (QY), concentration, incidence angle, polarization, and fluence rate F on imaging depth are comprehensively studied. Under F approved by the Food and Drug Administration, 1,300QDs with 50% QY can reach imaging depths of 29.7 mm in liver and 17.5 mm in skin. A time-gated excitation using 1,000 times higher F pulses can obtain the imaging depth of ≈ 5 cm. To validate our estimates, in vivo whole-body imaging experiments are performed using small-animal models.     

Aerenchyma Formed Under Phosphorus Deficiency Contributes to the Reduced Root Hydraulic Conductivity in Maize Roots

Root hydraulic conductivity has been shown to decrease under phosphorus （P） deficiency. This study Investigated how the formation of aerenchyma is related to this change. Root anatomy, as well as root hydraulic conductivity was studied In maize （Zea mays L.） roots under different phosphorus nutrition conditions. Plant roots under P stress showed enhanced degradation of cortical cells and the aerenchyma formation was associated with their reduced root hydraulic conductivity, supporting our hypothesis that air spaces that form in the cortex of phosphorusstressed roots Impede the radial transport of water in a root cylinder. Further evidence came from the variation In aerenchyma formation due to genotypic differences. Five maize inbred lines with different porosity in their root cortex showed a significant negative correlation with their root hydraulic conductivity. Shoot relative water content was also found lower In P-deficient maize plants than that in P-sufficient ones when such treatment was prolonged enough, suggesting a limitation of water transport due to lowered root hydraulic conductivity of P-deficient plants.     

Growth, Nitrogen Uptake and Flow in Maize Plants Affected by Root Growth Restriction

The objective of the present study was to investigate the influence of a reduced maize root-system size on root growth and nitrogen (N) uptake and flow within plants. Restriction of shoot-borne root growth caused a strong decrease in the absorption of root: shoot dry weight ratio and a reduction in shoot growth. On the other hand, compensatory growth and an increased N uptake rate in the remaining roots were observed. Despite the limited long-distance transport pathway in the mesocotyl with restriction of shoot-borne root growth, N cycling within these plants was higher than those in control plants, implying that xylem and phloem flow velocities via the mesocotyl were considerably higher than in plants with an intact root system. The removal of the seminal roots in addition to restricting shoot-borne root development did not affect whole plant growth and N uptake, except for the stronger compensatory growth of the primary roots. Our results suggest that an adequate N supply to maize plant is maintained by compensatory growth of the remaining roots, increased N uptake rate and flow velocities within the xylem and phloem via the mesocotyl, and reduction in the shoot growth rate.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Bioelectric and Morphological Response of Liquid-Covered Human Airway Epithelial Calu-3 Cell Monolayer to Periodic Deposition of Colloidal 3-Mercaptopropionic-Acid Coated CdSe-CdS/ZnS Core-Multishell Quantum Dots

Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs), are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER) of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods.     

Multi-Color Single Particle Tracking with Quantum Dots

Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.     

Electrophysiological responses of maize roots to low water potentials: relationship to growth and ABA accumulation

The maintenance of root elongation is an important adaptive response to low water potentials (psi(w)), but little is known about its regulation. An important component may be changes in root cell electrophysiology, which both signal and maintain growth maintenance processes. As a first test of this hypothesis, membrane potentials (E(m)) were measured within the cell elongation zone of maize (Zea mays L.) primary roots. Seedlings were grown in oxygenated solution culture, and low psi(w) was imposed by the gradual addition of polyethylene glycol. Cells hyperpolarized approximately 25 mV in response to low psi(w), and after 48 h resting potentials remained significantly hyperpolarized at psi(w) lower than -0.3 MPa compared with roots at high psi(w). Inhibitor experiments showed that the hyperpolarization was dependent on plasma membrane H(+)-ATPase activity. Previous work showed that accumulation of abscisic acid (ABA) is required for the maintenance of maize primary root elongation at low psi(w). To determine if the mechanism of action of ABA involves changes in root electrophysiology, E(m) measurements were made during long-term exposure to low psi(w). Steady-state resting E(m) were measured in regions in which maintenance of cell elongation was dependent on ABA accumulation (2-3 mm from the apex), or in which elongation was inhibited regardless of ABA status (6-8 mm from the apex). E(m) was substantially more negative in ABA-deficient roots specifically in the 2-3 mm region. The results suggest that set-points for ion homeostasis shifted in association with the maintenance of root cell elongation at low psi(w), and that ABA accumulation plays a role in regulating the ion transport processes involved in this response.     

Synthesis of a quinazoline derivative: a new α₁-adrenoceptor ligand for conjugation to quantum dots to study α₁-adrenoceptors in living cells

Quantum dots (QDs) that are conjugated to small molecule derivatives of drugs and endogenous ligands may be useful tools to study the distribution and dynamic of membrane bound receptors, ion channels and transporters in live cells. In order to use these tools, it is necessary to functionalize QDs with bioactive ligands. In this paper, we successfully synthesized a ligand of α(1)-adrenoceptor that could be conjugated to QDs. In addition, the conjugation of the ligands to QDs and their biological activity were evaluated through binding assay with 30 nM QD conjugates in living human embryonic kidney 293 cells.     

Respiratory potential of maize (Zea mays L.) roots exposed to hypoxia

When hypoxia is not too severe, root aerobic metabolism can be partly supported by oxygen delivery via aerenchymateous tissues. In terms of supplying energy, this adaptation is of special importance in plants with a high metabolic demand, such as maize (Zea mays L.). The ability of maize to respond to hypoxia by morphological changes is well documented; however, little is known on the potential for oxidative metabolism in different types of maize roots. In our study, we assessed the root respiratory potential in seminal and adventious nodal roots of maize exposed to mild short-term hypoxia. Plants responded to the treatment with an increased portion of nodal roots per total root length, while there were no changes in the biomass of shoots and roots. Thick nodal roots had much higher respiratory potential (Electron Transport System Activity – ETS) than nodal roots of smaller diameter or seminal roots, irrespective of the aeration rate. The only change in ETS under oxygen deficiency was found for seminal roots where oxygen consumption increased by 25%. Increased root porosity was observed in all roots, the increase was higher in nodal roots. On the basis of ETS data and taking into account changes of root morphology, it can be concluded that large changes of root respiratory potential are not involved in the response of maize to hypoxia. Obviously, maize can cover the respiratory needs with shifts in the growth of different root types which inherently differ in their potential aerobic respiration.     

Assembly and targeting of liposomal nanoparticles encapsulating quantum dots

Quantum dots (QDs) are attracting intense interest as fluorescence labeling agents for biomedical imaging because biocompatible coatings and relatively nontoxic rare earth metal QDs have emerged as possible options. QD photoemissions are bright, of narrow wavelength range, and very stable. We sought to encapsulate QDs within targeted PEGylated liposomes to reduce their propensity for liver uptake and to amplify the already strong QD emission signal. A novel lipid-QD conjugate initialized a process by which lipids in solution coalesced around the QDs. The liposomal structure was confirmed with size measurements, SEM, and IR spectroscopy. PEGylated QD liposomes injected into a xenograft tumor model largely cleared from the body within 24 h. Residual liver labeling was low. Targeted QD liposomes exhibited robust tumor labeling compared with controls. This study highlights the potential of these near IR emitting QD liposomes for preclinical/clinical applications.     

CdSe quantum dot (QD)-induced morphological and functional impairments to liver in mice

Quantum dots (QDs), as unique nanoparticle probes, have been used in in vivo fluorescence imaging such as cancers. Due to the novel characteristics in fluorescence, QDs represent a family of promising substances to be used in experimental and clinical imaging. Thus far, the toxicity and harmful health effects from exposure (including environmental exposure) to QDs are not recognized, but are largely concerned by the public. To assess the biological effects of QDs, we established a mouse model of acute and chronic exposure to QDs. Results from the present study suggested that QD particles could readily spread into various organs, and liver was the major organ for QD accumulation in mice from both the acute and chronic exposure. QDs caused significant impairments to livers from mice with both acute and chronic QD exposure as reflected by morphological alternation to the hepatic lobules and increased oxidative stress. Moreover, QDs remarkably induced the production of intracellular reactive oxygen species (ROS) along with cytotoxicity, as characterized by a significant increase of the malondialdehyde (MDA) level within hepatocytes. However, the increase of the MDA level in response to QD treatment could be partially blunted by the pre-treatment of cells with beta-mercaptoethanol (β-ME). These data suggested ROS played a crucial role in causing oxidative stress-associated cellular damage from QD exposure; nevertheless other unidentified mediators might also be involved in QD-mediated cellular impairments. Importantly, we demonstrated that the hepatoxicity caused by QDs in vivo and in vitro was much greater than that induced by cadmium ions at a similar or even a higher dose. Taken together, the mechanism underlying QD-mediated biological influences might derive from the toxicity of QD particles themselves, and from free cadmium ions liberated from QDs as well.     

Characterization of VCAM-1-Binding Peptide-Functionalized Quantum Dots for Molecular Imaging of Inflamed Endothelium

Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs) have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide) functionalized QDs (VQDs) from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor (TNF-) treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF--treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells'' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.     

Analysis method for measuring submicroscopic distances with blinking quantum dots

A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment.     

Accumulation and transport of nickel in relation to organic acids in ryegrass and maize grown with different nickel levels

Difference in Ni tolerance/accumulation in plant genotypes might be used to identify or develop plants for remediation of high Ni soils. Ryegrass was shown to be more sensitive to Ni toxicity and accumulated much more Ni in shoots than maize. The objectives of this study were to examine the relationship of organic acids to Ni accumulation and xylem transport of Ni in ryegrass (Lolium perenne L.) and maize (Zea mays L.). The results showed that accumulation of Ni in shoots was 5 to 7 fold higher in ryegrass than in maize grown at 20 to 80 µM Ni, whereas Ni concentration in ryegrass roots was only 1 to 2 fold higher at 0.1 to 40 µM Ni and 1.5-fold lower at 80 µM Ni than that of maize roots. Xylem transport rates of Ni increased with increasing Ni supply for both species, and were about 2 to 7 times higher in ryegrass than in maize. Shoot concentrations of citric, malic, oxalic and cis-aconitic acids increased at Ni levels above 20 µM, and were about 2 to 6 times higher in ryegrass than in maize. Whereas, maize roots accumulated greater amount of malic, oxalic, and cis-aconitic acids than ryegrass roots, especially at Ni levels of 40-80 µM. The rate of Ni exudation by roots in the two species was significantly correlated with root Ni concentrations. It could be concluded that high Ni accumulation in shoots was closely related to high xylem transport rates of Ni and that the accumulation of organic acids, citric and malic acid in particular. A high root exudate rate of Ni and the enhanced accumulation of organic acids, malic acid in particular, in roots might be among the important factors which are associated with the tolerance of crops to toxic Ni levels.     

Blinking effect and the use of quantum dots in single molecule spectroscopy

Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.