The aryl hydrocarbon receptor mediates sex ratio distortion in the embryos sired by TCDD-exposed male mice.

Many reports describe an association between preconceptional paternal exposure to environmental chemicals, including the persistent organic pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with an increased number of female offspring. We chronically treated wild-type C57BL/6 male mice with TCDD to investigate a role for the aryl hydrocarbon receptor (AHR) transcription factor. These mice had a 14 % lower male:female sex ratio than control mice, which was not observed in TCDD-treated Ahr knock out mice. AHR target genes Cyp1a1 and Ahrr were upregulated in the liver and testis of WT mice and Ahr expression was higher in the epididymis (2-fold) and liver (18-fold) than in whole testis tissue. The AHR protein was localized to round spermatids, elongating spermatids, and Leydig cells in the testis of WT mice. These studies demonstrate AHR involvement in the sex ratio distortion of TCDD-exposed males and the need for evaluating the molecular and genetic mechanism of this process.

Molecular Imaging of Mesothelin-Expressing Ovarian Cancer with a Human and Mouse Cross-Reactive Nanobody.

Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays. Initially, we performed microscale thermophoresis to determine the binding affinity between bNbG3a and human ( Kd = 46 ± 8 nM) or mouse ( Kd = 4.8 ± 0.4 nM) mesothelin protein. The human and mouse cross-reactivity was confirmed by in vivo optical imaging using bNbG3a bound to fluorescent streptavidin. We also localized the binding site of nNbG3a on human mesothelin using overlapping peptide scan. NbG3a recognized an epitope within residues 21-65 of the mature membrane bound form of human mesothelin, which is part of the N-terminal region of mesothelin that is important for interactions between mesothelin on peritoneal cells and CA125 on tumor cells. Next, the bNbG3a in vivo half-life after intravenous injection in healthy mice was estimated by ELISA assay to be 5.3 ± 1.3 min. In tumor-bearing animals, fluorescent bNbG3a accumulated in a subcutaneous ovarian xenograft (A1847) and in two syngeneic, orthotopic ovarian tumors (intraovary and intraperitoneal ID8) within an hour of intravenous injection that peaked by 4 h and persisted up to 48 h. MRI analysis of bNbG3a-targeted streptavidin-labeled iron oxides showed that the MRI signal intensity decreased 1 h after injection for a subcutaneous xenograft model of ovarian cancer for bNbG3a-labeled iron oxides compared to unlabeled iron oxides. The signal intensity differences continued up to the final time point at 24 h post injection. Finally, in vivo immunofluorescence 24 or 48 h after bNbG3a intravenous injection showed bNbG3a diffuse distribution of both xenograft and syngeneic ovarian tumors, with local areas of high concentration throughout A1847 human tumor. The data support the use of NbG3a for continued preclinical development and translation to human applications for cancers that overexpress mesothelin.

Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications.

Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems. We generated a phage-display library of Nbs that were amplified from B cells of a llama that was immunized with human recombinant mesothelin. Two nanobodies (Nb A1 and Nb C6) were selected on the basis of affinity (K(D) = 15 and 30 nM, respectively). Nb A1 was further modified by adding either a cysteine to permit maleimide-based bioconjugations or a sequence for the site-specific metabolic addition of a biotin in vivo. Both systems of conjugation (thiol-maleimide and streptavidin/biotin) were used to characterize and validate Nb A1 and to functionalize nanoparticles. We showed that anti-mesothelin Nb A1 could detect native and denatured mesothelin in various diagnostic applications, including flow cytometry, western blotting, immunofluorescence, and optical imaging. In conclusion, anti-mesothelin Nbs are novel, cost-effective, small, and single domain reagents with high affinity and specificity for the tumor-associated antigen mesothelin, which can be simply bioengineered for attachment to nanoparticles or modified surfaces using multiple bioconjugation strategies. These anti-mesothelin Nbs can be useful in both conventional and nanotechnology-based diagnostic, therapeutic and prognostic biomedical applications.

Biological barriers and current strategies for modifying nanoparticle bioavailability.

Nanoparticles are diligently crafted with exacting control over size, shape, and composition. The pristine nanoparticles are rigorously characterized in vitro by numerous physical and materials science techniques. Immediately after being exposed to body fluids, nanoparticles interact with a heterogeneous mixture of proteins and numerous different cell types that modify the nanoparticle surface and affect their bioavailability. Understanding the mechanisms behind the recognition and elimination of these modified nanoparticles is the key to the successful translation of nanomaterials from preclinical to clinical applications. This paper reviews the anatomy of the primary organs (kidney, liver, and spleen) responsible for nanoparticle bioelimination and the components of the innate immune system (complement system and scavenger receptors) that indirectly and directly recognize nanoparticles as foreign. Recent results using PEG as a steric barrier, generating biomimetic nanoparticles, and the effect of nanoparticle material properties to increase the bioavailability of nanoparticles are presented.

Facile immunotargeting of nanoparticles against tumor antigens using site-specific biotinylated antibody fragments.

Nanotechnology is actively being developed for preclinical and clinical oncology applications. Nanoparticle-based immunotargeting against tumor antigens with antibodies or antibody fragments is designed to increase the nanoparticle concentration at the tumor site. However, chemical-based strategies for bioconjugating antibody fragments to nanoparticles typically result in a functionally heterogeneous population of conjugates due to alteration of amino acids within the antigen binding site. The loss of function can be prevented by expressing recombinant antibodies that contain a unique bioconjugation site, which is isolated from the antigen binding site. Biobodies are antibody fragments biosynthetically biotinylated by yeast at a specific biotin acceptor site and secreted into the culture supernatant. The high specificity and affinity between streptavidin-labeled nanoparticles and soluble biobodies allow self-assembly of immunotargeted nanoparticles directly in the yeast culture supernatant. Here, we demonstrate the versatility of biobodies for nanoparticle immunotargeting using streptavidin-labeled superparamagnetic iron oxide nanoparticles as a general, modular scaffold. Biobody-mediated targeting was performed against two antigens (mesothelin and TEM1) that are upregulated in solid tumors. The technology for biosynthetic biotinylation can be extended to proteins other than antibody fragments and adopted by fields outside of oncology for directed modification of any streptavidin-functionalized surface.

Designing tripodal and triangular gadolinium oxide nanoplates and self-assembled nanofibrils as potential multimodal bioimaging probes.

Here, we report the shape-controlled synthesis of tripodal and triangular gadolinium oxide (Gd2O3) nanoplates. In the presence of lithium ions, the shape of the nanocrystals is readily controlled by tailoring reaction parameters such as temperature and time. We observe that the morphology transforms from an initial tripodal shape to a triangular shape with increasing reaction time or elevated temperatures. Highly uniform Gd2O3 nanoplates are self-assembled into nanofibril-like liquid-crystalline superlattices with long-range orientational and positional order. In addition, shape-directed self-assemblies are investigated by tailoring the aspect ratio of the arms of the Gd2O3 nanoplates. Due to a strong paramagnetic response, Gd2O3 nanocrystals are excellent candidates for MRI contrast agents and also can be doped with rare-earth ions to form nanophosphors, pointing to their potential in multimodal imaging. In this work, we investigate the MR relaxometry at high magnetic fields (9.4 and 14.1 T) and the optical properties including near-IR to visible upconversion luminescence and X-ray excited optical luminescence of doped Gd2O3 nanoplates. The complex shape of Gd2O3 nanoplates, coupled with their magnetic properties and their ability to phosphoresce under NIR or X-ray excitation which penetrate deep into tissue, makes these nanoplates a promising platform for multimodal imaging in biomedical applications.

Magnetization transfer induced biexponential longitudinal relaxation.

Longitudinal relaxation of brain water (1)H magnetization in mammalian brain in vivo is typically analyzed on a per-voxel basis using a monoexponential model, thereby assigning a single relaxation time constant to all (1)H magnetization within a given voxel. This approach was tested by obtaining inversion recovery (IR) data from gray matter of rats at 64 exponentially spaced recovery times. Using Bayesian probability for model selection, brain water data were best represented by a biexponential function characterized by fast and slow relaxation components. At 4.7T, the amplitude fraction of the rapidly relaxing component is 3.4% +/- 0.7% with a rate constant of 44 +/- 12 s(-1) (mean +/- SD; 174 voxels from four rats). The rate constant of the slow relaxing component is 0.66 +/- 0.04 s(-1). At 11.7T, the corresponding values are 6.9% +/- 0.9%, 19 +/- 5 s(-1), and 0.48 +/- 0.02 s(-1) (151 voxels from four rats). Several putative mechanisms for biexponential relaxation behavior were evaluated, and magnetization transfer (MT) between bulk water protons and nonaqueous protons was determined to be the source of biexponential longitudinal relaxation. MR methods requiring accurate quantification of longitudinal relaxation may need to take this effect explicitly into account.

Permeation peptide conjugates for in vivo molecular imaging applications.

Rapid and efficient delivery of imaging probes to the cell interior using permeation peptides has enabled novel applications in molecular imaging. Membrane permeant peptides based on the HIV-1 Tat basic domain sequence, GRKKRRQRRR, labeled with fluorophores and fluorescent proteins for optical imaging or with appropriate peptide-based motifs or macrocycles to chelate metals, such as technetium for nuclear scintigraphy and gadolinium for magnetic resonance imaging, have been synthesized. In addition, iron oxide complexes have been functionalized with the Tat basic domain peptides for magnetic resonance imaging applications. Herein we review current applications of permeation peptides in molecular imaging and factors influencing permeation peptide internalization. These diagnostic agents show concentrative cell accumulation and rapid kinetics and display cytosolic and focal nuclear accumulation in human cells. Combining methods, dual-labeled permeation peptides incorporating fluorescein maleimide and chelated technetium have allowed for both qualitative and quantitative analysis of cellular uptake. Imaging studies in mice following intravenous administration of prototypic diagnostic permeation peptides show rapid whole-body distribution allowing for various molecular imaging applications. Strategies to develop permeation peptides into molecular imaging probes have included incorporation of targeting motifs such as molecular beacons or protease cleavable domains that enable selective retention, activatable fluorescence, or targeted transduction. These novel permeation peptide conjugates maintain rapid translocation across cell membranes into intracellular compartments and have the potential for targeted in vivo applications in molecular imaging and combination therapy.

Synthesis and characterization of a Gd-DOTA-D-permeation peptide for magnetic resonance relaxation enhancement of intracellular targets.

Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 +/- 0.11 mM(-1) sec(-1) at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.

Selectivity in heavy metal- binding to peptides and proteins.

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.