Quantum dot/antibody conjugates for in vivo cytometric imaging in mice.

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1(+)c-Kit(+) cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.

Compact zwitterion-coated iron oxide nanoparticles for biological applications.

The potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various biomedical applications, including magnetic resonance imaging (MRI), sensing, and drug delivery, requires that their surface be derivatized to be hydrophilic and biocompatible. We report here the design and synthesis of a compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand with strong binding affinity to SPIONs. After ligand exchange, the ZDS-coated SPIONs exhibit small hydrodynamic diameters, and stability with respect to time, pH, and salinity. Furthermore, small ZDS coated SPIONs were found to have a reduced nonspecific affinity (compared to negatively charged SPIONs) toward serum proteins; streptavidin/dye functionalized SPIONs were bioactive and thus specifically targeted biotin receptors.

Electroluminescence from nanoscale materials via field-driven ionization.

The high degree of morphological and energetic disorder inherent to many nanosized materials places limitations on charge injection into and transport rates through thin films of these materials. We demonstrate electroluminescence achieved by local generation of charge that eliminates the need for injection of charge carriers from the device electrodes. We show electroluminescence from thin films of nanoscale materials that do not support direct current excitation and suggest a mechanism for the charge generation and electroluminescence that is consistent with our time-averaged and time-resolved observations.

Monovalent, reduced-size quantum dots for imaging receptors on living cells.

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

Air-stable operation of transparent, colloidal quantum dot based LEDs with a unipolar device architecture.

We report a novel unipolar light-emitting device architecture that operates using direct-current, field-driven electroluminescence of colloidally synthesized quantum dots (QDs). This device architecture, which is based only on transparent ceramics and QDs, enables emission from different color QDs and, for the first time, constant QD electroluminescence during extended operation in air, unpackaged.

InAs(ZnCdS) quantum dots optimized for biological imaging in the near-infrared.

We present the synthesis of InAs quantum dots (QDs) with a ZnCdS shell with bright and stable emission in the near-infrared (NIR, 700-900 nm) region for biological imaging applications. We demonstrate how NIR QDs can image tumor vasculature in vivo at significantly deeper penetration depths and with higher contrast than visible emitting CdSe(CdS) QDs. Targeted cellular labeling is also presented and may enable multiplexed and low autofluorescence cellular imaging.

Compact biocompatible quantum dots via RAFT-mediated synthesis of imidazole-based random copolymer ligand.

We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (approximately 10-12 nm diameter), high quantum yields (>50%), excellent stability across a large pH range (pH 5-10.5), and low nonspecific binding. To address the fundamental problem of thiol instability in traditional ligand exchange systems, the polymers here employ a stable multidentate imidazole binding motif to the QD surface. The polymers are synthesized via reversible addition-fragmentation chain transfer-mediated polymerization to produce molecular weight controlled monodisperse random copolymers from three types of monomers that feature imidazole groups for QD binding, polyethylene glycol (PEG) groups for water solubilization, and either primary amines or biotin groups for derivatization. The polymer architecture can be tuned by the monomer ratios to yield aqueous QDs with targeted surface functionalities. By incorporating amino-PEG monomers, we demonstrate covalent conjugation of a dye to form a highly efficient QD-dye energy transfer pair as well as covalent conjugation to streptavidin for high-affinity single molecule imaging of biotinylated receptors on live cells with minimal nonspecific binding. The small size and low serum binding of these polymer-coated QDs also allow us to demonstrate their utility for in vivo imaging of the tumor microenvironment in live mice.

Tissue- and organ-selective biodistribution of NIR fluorescent quantum dots.

A significant portion of the field of nanomedicine is predicated on being able to target nanoparticles to sites of disease. However, in vivo biodistribution and clearance of nanoparticles are poorly understood. In this study, a novel formulation of near-infrared fluorescent InAs(ZnS) quantum dots was synthesized and coated with a systematically increasing chain length of PEG. We found that varying PEG chain length resulted in major changes in organ/tissue-selective biodistribution and clearance from the body.

Monodisperse and Water-Soluble Quantum Dots for SWIR Imaging via Carboxylic Acid Copolymer Ligands.

Compared to the visible and near-infrared, the short-wave infrared region (SWIR; 1000-2000 nm) has excellent properties for in vivo imaging: low autofluorescence, reduced scattering, and a low-absorption cross-section of blood or tissue. However, the general adoption of SWIR imaging in biomedical research will be enhanced by a broader availability of versatile and bright contrast materials. Quantum dots (QDs) are bright and compact SWIR emitters with narrow size distributions and emission spectra, but their use is limited by the shortcomings of established ligand systems for SWIR QDs. Established ligands often result in SWIR probes with either limited colloidal stability, large size, or broad size distribution or a combination of all three. We present a polymeric QD ligand designed to be compatible with oleate-coated QDs. Our polymeric acid ligand is a copolymer bearing carboxylic acid anchoring groups and PEG-550 chains to solubilize the QD-ligand construct. After a mild and rapid ligand exchange, the resulting constructs are compact (<11 nm hydrodynamic diameter) and have narrow size distribution. Both qualities are preserved for several months in isotonic saline. The constructs are bright in vivo, and to demonstrate their suitability for imaging, we perform whole-body imaging and lymphatic imaging, including visualization of lymphatic flow.

Compact biocompatible quantum dots functionalized for cellular imaging.

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor's local chemical environment.

Compact zwitterion-coated iron oxide nanoparticles for in vitro and in vivo imaging.

We have recently developed compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand coated superparamagnetic iron oxide nanoparticles (SPIONs) for use in various biomedical applications. The defining characteristics of ZDS-coated SPIONs are small hydrodynamic diameters, low non-specific interactions with fetal bovine serum, the opportunity for specific labeling, and stability with respect to time, pH, and salinity. We report here on the magnetic characterization of ZDS-coated SPIONs and their in vitro and in vivo performance relative to non-specific interactions with HeLa cells and in mice, respectively. ZDS-coated SPIONs retained the superparamagnetism and saturation magnetization (M(s)) of as-synthesized hydrophobic SPIONs, with M(s) = 74 emu g(-1) [Fe]. Moreover, ZDS-coated SPIONs showed only small non-specific uptake into HeLa cancer cells in vitro and low non-specific binding to serum proteins in vivo in mice.

In-situ encapsulation of quantum dots into polymer microspheres.

We have incorporated fluorescent quantum dots (QDs) into polystyrene microspheres using functionalized oligomeric phosphine (OP) ligands. We find that a uniform distribution of quantum dots is loaded inside each polymer bead. Some local close-packing of quantum dots in the beads is attributed to the self-polymerization of the functionalized ligands. The presence of quantum dots disturbs the nucleation and growth processes during the formation of polymer microspheres and results in a wider size distribution of the quantum dot-embedded polystyrene beads than for the control without dots. The change in quantum efficiency of the quantum dots before (approximately 20%) and after (12%) loading into the beads substantiates the protection of oligomeric phosphine ligands yet indicates that the properties of these quantum dots are still affected during processing.

Size series of small indium arsenide-zinc selenide core-shell nanocrystals and their application to in vivo imaging.

We have developed a size series of unusually small, water-soluble (InAs)ZnSe (core)shell quantum dots (QDs) that emit in the near-infrared and exhibit new behavior in vivo, including multiple sequential lymph node mapping and extravasation from the vasculature. The biological utility of these fluorescent probes resulted from our intentional choice to match the semiconductor material and water-soluble ligand with a desired final hydrodynamic diameter and emission wavelength.

Phosphine oxide polymer for water-soluble nanoparticles.

A phosphine oxide polymer was developed using bis(dichlorophosphino)ethane and poly(ethylene glycol). This polymer system was used to transfer various nanoparticles from organic solvents to water, retaining their physical properties and reactivities.

Engineering InAs(x)P(1-x)/InP/ZnSe III-V alloyed core/shell quantum dots for the near-infrared.

Quantum dots with a core/shell/shell structure consisting of an alloyed core of InAs(x)P(1-x), an intermediate shell of InP, and an outer shell of ZnSe were developed. The InAs(x)P(1-x) alloyed core has a graded internal composition with increasing arsenic content from the center to the edge of the dots. This compositional gradient results from two apparent effects: (1) the faster reaction kinetics of the phosphorus precursor compared to the arsenic precursor, and (2) a post-growth arsenic-phosphorus exchange reaction that increases the arsenic content. The cores have a zinc blend structure for all compositions and show tunable emission in the near-infrared (NIR) region. A first shell of InP leads to a red-shift and an increase in quantum yield. The final shell of ZnSe serves to stabilize the dots for applications in aqueous environments, including NIR biomedical fluorescence imaging. These NIR-emitting core/shell/shell InAs(x)P(1-x)/InP/ZnSe were successfully used in a sentinel lymph node mapping experiment.