In Vivo Pretargeted Imaging of HER2 and TAG-72 Expression Using the HaloTag Enzyme.

A novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands In-HTL-1, -2, and -3 were evaluated as secondary agents.

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag.

The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells.

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets.

Development of an enzymatic pretargeting strategy for dual-modality imaging.

A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.

Dermatitis herpetiformis: pathophysiology, clinical presentation, diagnosis and treatment.

Researches on DH have shown that it is not just a bullous skin disease, but a cutaneous-intestinal disorder caused by hypersensitivity to gluten. Exposure to gluten is the starting point of an inflammatory cascade capable of forming autoantibodies that are brought to the skin, where they are deposited, culminating in the formation of skin lesions. These lesions are vesico-bullous, pruritic, and localized especially on elbows, knees and buttocks, although atypical presentations can occur.

HaloTag as a reporter gene: positron emission tomography imaging with (64)Cu-labeled second generation HaloTag ligands.

THE GOAL OF THIS STUDY IS TO EMPLOY THE HALOTAG TECHNOLOGY FOR POSITRON EMISSION TOMOGRAPHY (PET), WHICH INVOLVES TWO COMPONENTS: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N'N''-triacetic acid).

Pyodermitis.

Pyodermitis are primary skin infections mainly caused by pyogenic bacteria of the Staphylococcus and Streptococcus genera. They are relatively common diseases that affect adults and children. There have been frequent reports of bacterial resistance to the recommended antibiotics over the last few years; however, new substances are in use or under development, and this represents an evolution in the treatment of pyodermitis.

HaloTag: a novel reporter gene for positron emission tomography.

Among the many molecular imaging techniques, reporter gene imaging has been a dynamic area of research. The HaloTag protein is a modified haloalkane dehalogenase which was designed to covalently bind to synthetic ligands (i.e.

Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition.

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes.

Design of biotin-functionalized luminescent quantum dots.

We report the design and synthesis of a tetraethylene glycol- (TEG-) based bidentate ligand functionalized with dihydrolipoic acid (DHLA) and biotin (DHLA-TEG-biotin) to promote biocompatibility of luminescent quantum dots (QD's). This new ligand readily binds to CdSe-ZnS core-shell QDs via surface ligand exchange. QDs capped with a mixture of DHLA and DHLA-TEG-biotin or polyethylene glycol- (PEG-) (molecular weight average approximately 600) modified DHLA (DHLA-PEG600) and DHLA-TEG-biotin are easily dispersed in aqueous buffer solutions.

The effect of molecular orientation on the potential of porphyrin-metal contacts.

The effect of molecular orientation at metal contacts on interface properties was determined by examining the local work function of porphyrin on atomically smooth graphite. The orientation was varied by self-assembly from the vapor phase, and the local potential was quantified by Kelvin force microscopy (scanning surface potential microscopy). When the porphyrin ring is oriented parallel to the substrate, the surface work function is 50 mV less than that of the highly ordered pyrolytic graphite; in contrast, when the porphyrin molecular plane is oriented perpendicular to the substrate, the surface work function is unchanged.

Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands.

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps.

Luminescent biocompatible quantum dots: a tool for immunosorbent assay design.

We have developed several conjugation strategies based on noncovalent self-assembly for the attachment of proteins and other biomolecules to water-soluble luminescent colloidal semiconductor nanocrystals (quantum dots [QDs]). The resulting QD-protein conjugates were employed in designing a variety of bioinspired applications, including single and multiplexed immunosorbent assays to detect toxins and small molecule explosives. In these studies we showed that QD fluorophores offer several important advantages.

Hydrodynamic dimensions, electrophoretic mobility, and stability of hydrophilic quantum dots.

Luminescent semiconductor quantum dots (QDs) have great potential for use in biological assays and imaging. These nanocrystals are capped with surface ligands (bifunctional molecules, amphiphilic polymers, phospholipids, etc.) that render them hydrophilic and provide them with functional properties.

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types.

Quantum dot-based multiplexed fluorescence resonance energy transfer.

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured.

Broad spectral domain fluorescence wavelength modulation of visible and near-infrared emissive polymersomes.

Incorporation of an extended family of multi[(porphinato)zinc(II)] (PZn)-based supermolecular fluorophores into the lamellar membranes of polymersomes (50 nm to 50 mum diameter polymer vesicles) gives rise to electrooptically diverse nano-to-micron (meso) scale soft materials. Studies that examine homogeneous suspensions of 100 nm diameter emissive polymersomes demonstrate fluorescence energy modulation over a broad spectral domain of the visible and near-infrared (600-900 nm). These polymersomal structures highlight that the nature of intermembranous polymer-to-fluorophore contacts depends on the position and identity of the porphyrins' phenyl ring substituents.

Quantum dot bioconjugates for imaging, labelling and sensing.

One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations, in which traditional fluorescent labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water soluble and target them to specific biomolecules has led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as efficient fluorescence resonance energy transfer donors.

A hybrid quantum dot-antibody fragment fluorescence resonance energy transfer-based TNT sensor.

We demonstrate the use of luminescent QDs conjugated to antibody fragments to develop solution-phase nanoscale sensing assemblies, based on fluorescence resonance energy transfer (FRET) for the specific detection of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments. The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue prebound in the antibody binding site quenches the QD photoluminescence via proximity-induced FRET.

Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores.

We describe a simple and versatile scheme to prepare an array of heterofunctional multidentate ligands that permit strong and stable interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and render them soluble in aqueous environments. These ligands were synthesized by reacting various chain length poly(ethylene glycols) with thioctic acid, followed by ring opening of the dithiolane moiety, creating a bidentate thiol motif with enhanced affinity for CdSe-ZnS core-shell QDs. Functionalization with these ligands permits processability of the nanocrystals not only in aqueous but also in many other polar solvents.

Surface-immobilized self-assembled protein-based quantum dot nanoassemblies.

Luminescent semiconductor quantum dot (QD)-based optical biosensors have the potential to overcome many of the limitations associated with using conventional organic dyes for biodetection. We have previously demonstrated a hybrid QD-protein-based fluorescence resonance energy transfer (FRET) sensor. Although the QD acted as an energy donor and a protein scaffold in the sensor, recognition and specificity were derived from the proteins.

Highly conjugated (polypyridyl)metal-(porphinato)zinc(II) compounds: long-lived, high oscillator strength, excited-state absorbers having exceptional spectral coverage of the near-infrared.

Transient dynamical studies of ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)2+ bis-hexafluorophosphate (Ru-PZn), osmium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)2+ bis-hexafluorophosphate (Os-PZn), ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-15-(4'-nitrophenyl)ethynyl-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)2+ bis-hexafluorophosphate (Ru-PZn-A), osmium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-15-(4'-nitrophenyl)ethynyl-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)2+ bis-hexafluorophosphate (Os-PZn-A), and ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-ruthenium(II)-15-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-bis(2,2';6',2' '-terpyridine)4+ tetrakis-hexafluorophosphate (Ru-PZn-Ru), and ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-osmium(II)-15-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-bis(2,2';6',2' '-terpyridine) tetrakis-hexafluorophosphate (Ru-PZn-Os) show that these highly conjugated supermolecular chromophores feature electronically excited states that absorb over broad NIR spectral windows with considerable oscillator strength and manifest lifetimes (1-50 mus) that are extraordinarily long relative to those of classic low band-gap organic materials. The excited-state absorptive domains of these strongly coupled multipigment ensembles can be extensively modulated. For sequential one-photon absorptive processes, these compounds evince large sigmae, sigmae/sigmag, and sigmae - sigmag values.

Multiplexed toxin analysis using four colors of quantum dot fluororeagents.

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate.

Unusual frequency dispersion effects of the nonlinear optical response in highly conjugated (polypyridyl)metal-(porphinato)zinc(II) chromophores.

The syntheses and electrooptic properties of a new family of nonlinear optical chromophores are reported. These species feature an ethyne-elaborated, highly polarizable porphyrinic component and metal polypyridyl complexes that serve as integral donor and acceptor elements. Examples of this structural motif include ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)(2+) bis-hexafluorophosphate (Ru-PZn); osmium(II) [5-(4'-ethynyl-(2,2';6',2''-terpyridinyl))-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2''-terpyridine)(2+) bis-hexafluorophosphate (Os-PZn); ruthenium(II) [5-(4'-ethynyl-(2,2';6',2''-terpyridinyl))-15-(4'-nitrophenyl)ethynyl-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phen-yl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)(2+) bis-hexafluorophosphate (Ru-PZn-A); osmium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))-15-(4'-nitrophenyl)ethynyl-10,20-bis(2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2';6',2' '-terpyridine)(2+) bis-hexafluorophosphate (Os-PZn-A); and ruthenium(II) [5-(4'-ethynyl-(2,2';6',2' '-terpyridinyl))osmium(II)-15-(4'-ethynyl-(2,2';6',2''-terpyridinyl))-10,20-bis (2',6'-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-bis(2,2';6',2''-terpyridine)(4+) tetrakis-hexafluorophosphate (Ru-PZn-Os).