13 research outputs found
Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications.
No full textInternational audienceMesothelin, 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
Coating Evaluation and Purification of Monodisperse, Water-Soluble, Magnetic Nanoparticles Using Sucrose Density Gradient Ultracentrifugation
No full textDesigning Tripodal and Triangular Gadolinium Oxide Nanoplates and Self-Assembled Nanofibrils as Potential Multimodal Bioimaging Probes
No full textSynthesis and Characterization of a Gd-DOTA-D-Permeation Peptide for Magnetic Resonance Relaxation Enhancement of Intracellular Targets
No full textSynthesis and Characterization of a Gd-DOTA--Permeation Peptide for Magnetic Resonance Relaxation Enhancement of Intracellular Targets
No full textMany 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
Coating Evaluation and Purification of Monodisperse, Water-Soluble, Magnetic Nanoparticles Using Sucrose Density Gradient Ultracentrifugation
No full textCoating Evaluation and
Purification of Monodisperse,
Water-Soluble, Magnetic Nanoparticles Using Sucrose Density Gradient
Ultracentrifugatio
Molecular Imaging of Mesothelin-Expressing Ovarian Cancer with a Human and Mouse Cross-Reactive Nanobody
No full textPermeation Peptide Conjugates for In Vivo Molecular Imaging Applications
No full textRapid 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
Designing Tripodal and Triangular Gadolinium Oxide Nanoplates and Self-Assembled Nanofibrils as Potential Multimodal Bioimaging Probes
No full textHere, 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
Molecular Imaging of Mesothelin-Expressing Ovarian Cancer with a Human and Mouse Cross-Reactive Nanobody
No full textMesothelin
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
