Manganese-based nanoscale metal-organic frameworks for magnetic resonance imaging

Manganese-containing nanoscale metal-organic frameworks (NMOFs) with controllable morphologies were synthesized using reverse-phase microemulsion techniques at room temperature and a surfactant-assisted procedure at 120 degrees C with microwave heating. The nanoparticles were characterized using a variety of methods including SEM, TEM, TGA, PXRD, and ICP-MS. Although the nanoparticles gave a modest longitudinal relaxivity (r1) on a per Mn basis, they provided an efficient vehicle for the delivery of large doses of Mn2+ ions which exhibited very high in vitro and in vivo r1 values and afforded excellent MR contrast enhancement. The particle surface was also modified with a silica shell to allow covalent attachment of a cyclic RGD peptide and an organic fluorophore. The cell-targeting molecules on the Mn NMOFs enhanced their delivery to cancer cells to allow for target-specific MR imaging in vitro. The MR contrast enhancement was also demonstrated in vivo using a mouse model. Such core-shell hybrid nanostructures provide an ideal platform for targeted delivery of other imaging and therapeutic agents to diseased tissues.     

Synthesis and Evaluation of GdIII‐Based Magnetic Resonance Contrast Agents for Molecular Imaging of Prostate‐Specific Membrane Antigen

Magnetic resonance (MR) imaging is advantageous because it concurrently provides anatomic, functional, and molecular information. MR molecular imaging can combine the high spatial resolution of this established clinical modality with molecular profiling in vivo. However, as a result of the intrinsically low sensitivity of MR imaging, high local concentrations of biological targets are required to generate discernable MR contrast. We hypothesize that the prostate‐specific membrane antigen (PSMA), an attractive target for imaging and therapy of prostate cancer, could serve as a suitable biomarker for MR‐based molecular imaging. We have synthesized three new high‐affinity, low‐molecular‐weight Gd^III‐based PSMA‐targeted contrast agents containing one to three Gd^III chelates per molecule. We evaluated the relaxometric properties of these agents in solution, in prostate cancer cells, and in an in vivo experimental model to demonstrate the feasibility of PSMA‐based MR molecular imaging.     

A Structural Basis for 129Xe Hyper-CEST Signal in TEM-1 β-Lactamase

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized ¹²⁹Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 β-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.     

Basic design and synthesis of sulfonated contrast agents for magnetic resonance imaging based on gadolinium complexes.

Magnetic resonance angiography (MRA) is an imaging method to examine blood vessels based on the magnetic resonance imaging (MRI) technique. For this purpose, blood pool contrast agents have been developed to selectively increase the signal intensity of the intravascular lumen for improvement of the contrast-to-noise ratio in MR images. Here, we describe the design and the syntheses of six novel sulfonated contrast agents (KMR-Sulfo1 - 6), their chemical properties and their in vivo applications. In this study, we investigated the lipophilicity and the hydrophilicity of a gadolinium complex using a convenient two-step synthesis route, with the goal of prolonging the plasma half-life by binding mainly to human serum albumin. We confirmed that KMR-Sulfo5 fulfilled the requirements as a blood pool contrast agent: it showed a sufficient relaxivity r(1) of 5.9 mM(-1) s(-1), a long plasma half-life of 25.7 min and complete elimination from the body within 12 h after the administration.     

Nanoshell magnetic resonance imaging contrast agents

Nanocontrast agents have great potential in magnetic resonance (MR) molecular imaging applications for clinical diagnosis. We synthesized Au(3)Cu(1) (gold and copper) nanoshells that showed a promising MR contrast effect. For in vitro MR images, the large proton r1 relaxivities brightened T(1)-weighted images. As for the proton-dephasing effect in T(2), Au(3)Cu(1) lightened MR images at the low concentration of 0.125 mg mL(-1) (3.84 x 10(-7) mM), and then the signal continuously decreased as the concentration increased. For in vivo MR imaging, Au(3)Cu(1) nanocontrast agents enhanced the contrast of blood vessels and suggested their potential use in MR angiography as blood-pool agents. We propose that (1) the cooperativity originating from the form of the nanoparticles and (2) the large surface area coordinated to water from their porous hollow morphology are important for efficient relaxivity. In a cytotoxicity and animal survival assay, Au(3)Cu(1) nanocontrast agents showed a dose-dependent toxic effect: the viability rate of experimental mice reached 83% at a dose of 20 mg kg(-1) and as much as 100% at 2 mg kg(-1).     

A cell-penetrating protein designed for bimodal fluorescence and magnetic resonance imaging

Multimodal imaging is a highly desirable biomedical application since it can provide complementary information from each imaging modality. We propose a protein engineering-based strategy for the construction of a bimodal probe for fluorescence and magnetic resonance imaging. A recombinant protein was generated by the fusion of a supercharged green fluorescence protein (GFP³⁶⁺) with a lanthanide-binding tag (dLBT) that can stably bind two Gd³⁺ ions. The GFP³⁶⁺–dLBT fusion protein showed strong fluorescence and exhibited efficient contrast enhancement in magnetic resonance imaging. This protein probe improves the MR relaxation more efficiently than Gd-DTPA (gadopentetate dimeglumine). The superior cell-penetrating activity of GFP³⁶⁺ allows the efficient cellular uptake of this fusion protein and it can thus be used as a cellular imaging probe. Dual imaging was conducted in vitro and in mice. This result indicates that the fusion of different functional domains is a feasible approach for making multi-modal imaging agents.     

Sensitive protein microarray synergistically amplified by polymer brush-enhanced immobilizations of both probe and reporter

Great challenge remains to continuously improve sensitivity of protein microarrays for broad applications. A copolymer brush is in situ synthesized on both substrate and silica nanoparticle (SNP) surface to efficiently immobilize probe and reporter protein respectively for synergistic amplification of protein microarray signals. As a demonstration, sandwich immunoassay for a cancer biomarker carcinoembryonic antigen (CEA) detection is performed on microarray platform, showing a limit of detection (LOD) of 10 pg/ml and dynamic range of 10 pg/ml to 100 ng/ml. Two orders improvement of LOD is achieved in comparison to the small crosslinker-activated substrate. The improved sensitivity is attributed to not only the high immobilization amount of both probe and reporter but also the favorite protein binding orientations offered by the flexible brushes. This work provides a universal approach to inexpensively and significantly improve protein microarray sensitivity.     

Design and synthesis of an enzyme activity-based labeling molecule with fluorescence spectral change

Methods of covalent labeling of a specific tag protein with small-molecular dyes play an important role in studying dynamic behaviors of proteins in living cells. On the basis of quinone methide chemistry, we designed and synthesized a beta-galactosidase labeling probe, CMFbeta-gal, which shows a fluorescence wavelength change accompanying the labeling reaction, owing to fluorescence resonance energy transfer (FRET). Since the FRET efficiency changes accompanying the labeling reaction, fluorescence of labeled protein can be observed separately from that of the unreacted probe, so immediate detection of the target protein is possible. This is the first report of a protein labeling probe which features a change of fluorescence wavelength upon reaction, allowing the labeled protein to be detected even in the presence of unreacted probe.     

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.     

A sensitive and label-free impedimetric biosensor based on an adjunct probe

A highly sensitive and label-free impedimetric biosensor is achieved based on an adjunct probe attached nearby the capture probe. In this work, the adjunct probe was co-assembled on the surface of gold electrode with the capture probe hybridized with the reporter probe, and then 6-mercapto-1-hexanol was employed to block the nonspecific binding sites. When target DNA was added, the adjunct probe functioned as a fixer to immobilize the element of reporter probe displaced by the target DNA sequences and made the reporter probe approach the electrode surface, leading to effective inhibition of charge transfer. The increase in charge transfer resistance is related to the quantity of the target DNA in a wide range. The linear range for target DNA with specific sequences was from 0.1 nM to 0.5 μM with a good linearity (R = 0.9988) and a low detection limit of 6.3 pM. This impedimetric biosensor has the advantages of simplicity, sensitivity, good selectivity, and large dynamic range.     

Rational design of protein-based MRI contrast agents

We describe the rational design of a novel class of magnetic resonance imaging (MRI) contrast agents with engineered proteins (CAi.CD2, i = 1, 2,..., 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high-coordination Gd(3+) binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd(3+) over physiological metal ions such as Ca(2+), Zn(2+), and Mg(2+). These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small-molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, thereby extending applications of MRI.     

A Dansyl Amide N-Oxide Fluorogenic Probe Based on a Bioorthogonal Decaging Reaction

A smart fluorescence “turn-on” probe which contained a dansyl amide fluorophore and an N-oxide group was designed based on the bioorthogonal decaging reaction between N-oxide and the boron reagent. The reaction proceeds in a rapid kinetics (k₂=57.1±2.5 m ⁻¹ s⁻¹), and the resulting reduction product showcases prominent fluorescence enhancement (up to 72-fold). Time dependent density functional theoretical (TD-DFT) calculation revealed that the process of photoinduced electron transfer (PET) from the N-oxide moiety to the dansyl amide fluorophore accounts for the quenching mechanism of N-oxide. This probe also showed high selectivity over various nucleophilic amino acids and good biocompatibility in physiological conditions. The successful application of the probe in HaloTag protein labeling and HepG2 live-cell imaging proves it a valuable tool for visualization of biomolecules.     

Radiolabeled peptide probe for tumor imaging

Radionuclide imaging is now the premier imaging method in clinical practice for its high sensitivity and tomographic capability. Current clinically available radio imaging methods mostly use positron-emission tomography (PET) and single-photon emission computed tomography (SPECT) to detect anatomic abnormalities that conventional imaging techniques typically have challenges for visualizing. Contrast agents are indispensable for radionuclide imaging, and the radionuclide is always attached to a suitable vector that achieves targeted delivery. Nowadays, peptides have attracted increasing interest in targeting vectors of contrast agents, mainly due to their high specificity for target receptors at nanomolar concentrations and low toxicity. Radiolabeled peptide probes as kinds of PET/SPECT tracers had become essential tools for clinical radionuclide diagnosis. This review mainly summarizes radiolabeled peptide probes for bioimaging, including fundamental concepts of radiolabeled peptide probe design, some typical peptide analogs radiocontrast agents for PET, SPECT, and the combination imaging.     

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.     

Hybrid molecular probe for nucleic acid analysis in biological samples

The ability to detect changes in gene expression, especially in real-time and with sensitivity sufficient enough to monitor small variations in a single-cell, will have considerable value in biomedical research and applications. Out of the many available molecular probes for intracellular monitoring of nucleic acids, molecular beacon (MB) is the most frequently used probe with the advantages of high sensitivity and selectivity. However, any processes in which the MB stem-loop structure is broken will result in a restoration of the fluorescence in MB. This brings in a few possibilities for false positive signal such as nuclease degradation, protein binding, thermodynamic fluctuation, solution composition variations (such as pH, salt concentration) and sticky-end pairing. These unwanted processes do exist inside living cells, making nucleic acid monitoring inside living cells difficult. We have designed and synthesized a hybrid molecular probe (HMP) for intracellular nucleic acid monitoring to overcome these problems. HMP has two DNA probes, one labeled with a donor and the other an acceptor. The two DNA probes are linked by a poly(ethylene glycol) (PEG) linker, with each DNA being complementary to adjacent areas of a target sequence. Target binding event brings the donor and acceptor in proximity, resulting in quenching of the donor fluorescence and enhancement of the acceptor emission. The newly designed HMP has high sensitivity, selectivity, and fast hybridization kinetics. The probe is easy to design and synthesize. HMP does not generate any false positive signal upon digestion by nuclease, binding by proteins, forming complexes by sticky-end pairing, or by other molecular interaction processes. HMP is capable of selectively detecting nucleic acid targets from cellular samples.     

Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.     

One-pot chemo-enzymatic synthesis of reporter-modified proteins

To meet recent advancements in the covalent reporter labeling of proteins, we propose a flexible synthesis for reporter analogs. Here we demonstrate a one-pot chemo-enzymatic synthesis of reporter-labeled proteins that allows the covalent tethering of any amine-terminal fluorescent or affinity label to a carrier protein or fusion construct. This two-reaction sequence consists of activated panthothenate coupling, biosynthetic conversion to the coenzyme A (CoA) analog, and enzymatic carrier protein modification via phosphopantetheinyltransferase (PPTase). We also probe substrate specificity for CoAA, the first enzyme in the pathway. With this approach CoA analogs may be rapidly prepared, thus permitting the regiospecific attachment of reporter moieties from a variety of molecular species.     

A new class of macrocyclic lanthanide complexes for cell labeling and magnetic resonance imaging applications

Lanthanide complexes have wide applications in biochemical research and biomedical imaging. We have designed and synthesized a new class of macrocyclic lanthanide chelates, Ln/DTPA-PDA-C(n), for cell labeling and magnetic resonance imaging (MRI) applications. Two lipophilic Gd3+ complexes, Gd/DTPA-PDA-C(n) (n = 10, 12), labeled a number of cultured mammalian cells noninvasively at concentrations as low as a few micromolar. Cells took up these agents rapidly and showed robust intensity increases in T1-weighed MR images. Labeled cells showed normal morphology and doubling time as control cells. In addition to cultured cells, these agents also labeled primary cells in tissues such as dissected pancreatic islets. To study the mechanism of cellular uptake, we applied the technique of diffusion enhanced fluorescence resonance energy transfer (DEFRET) to determine the cellular localization of these lipophilic lanthanide complexes. After loading cells with a luminescent complex, Tb/DTPA-PDA-C10, we observed DEFRET between the Tb3+ complex and extracellular, but not intracellular, calcein. We concluded that these cyclic lanthanide complexes label cells by inserting two hydrophobic alkyl chains into cell membranes with the hydrophilic metal binding site facing the extracellular medium. As the first imaging application of these macrocyclic lanthanide chelates, we labeled insulin secreting beta-cells with Gd/DTPA-PDA-C12. Labeled cells were encapsulated in hollow fibers and were implanted in a nude mouse. MR imaging of implanted beta-cells showed that these cells could be followed in vivo for up to two weeks. The combined advantages of this new class of macrocyclic contrast agents ensure future imaging applications to track cell movement and localization in different biological systems.