Harald Kratz

Synthesis of acid-stabilized iron oxide nanoparticles and comparison for targeting atherosclerotic plaques: evaluation by MRI, quantitative MPS, and TEM alternative to ambiguous Prussian blue iron staining.

UNLABELLED To further optimize citrate-stabilized VSOPs (very small iron oxide particles, developed for MR angiography) for identification of atherosclerotic plaques, we modified their surface during synthesis using eight other acids for electrostatic stabilization. This approach preserves effective production for clinical application. Five particles were suitable to be investigated in targeting plaques of apoE(-/-) mice. Accumulation was evaluated by ex vivo MRI, TEM, and quantitatively by magnetic particle spectroscopy (MPS). Citric- (VSOP), etidronic-, tartaric-, and malic-acid-coated particles accumulated in atherosclerotic plaques with highest accumulation for VSOP (0.2‰ of injected dose). Targets were phagolysosomes of macrophages and of altered endothelial cells. In vivo MRI with VSOP allowed for definite plaque identification. Prussian blue staining revealed abundant endogenous iron in plaques, indistinguishable from particle iron. In apoE(-/-) mice, VSOPs are still the best anionic iron oxide particles for imaging atherosclerotic plaques. MPS allows for quantification of superparamagnetic nanoparticles in such small specimens. FROM THE CLINICAL EDITOR The presence of vulnerable plaques in arteries is important for the prediction of acute coronary events. VSOP (very small iron oxide particles, developed for MR angiography) have been shown to be very sensitive in identifying atherosclerotic plaques. The authors studied here further modification to the surface of VSOP during synthesis and compared their efficacy.

XTEN-annexin A5: XTEN allows complete expression of long-circulating protein-based imaging probes as recombinant alternative to PEGylation.

The coupling of polyethylene glycol (PEG) to proteins (PEGylation) has become a standard method to prolong blood circulation of imaging probes and other proteins, liposomes, and nanoparticles. However, concerns have arisen about the safety of PEG, especially with respect to its poor biodegradability and antibody formation, including new evidence about preformed anti-PEG antibodies in a quarter of healthy blood donors. Here, we apply a new hydrophilic polypeptide XTEN to extend the blood half-life of an imaging probe. As an example, we chose annexin A5 (AnxA5), a recombinant 35-kD protein extensively used for the in vitro and in vivo detection of apoptosis, that has a blood half-life of less than 7 min in mice, limiting its accumulation in target tissues and therefore limiting its utility as an imaging reagent. Methods: The sequence of XTEN was developed by Volker Schellenberger and colleagues by evolutionary in vitro optimization to yield PEG-like properties but provides several key advantages in comparison to PEG. The DNA of a 288-amino-acid version of XTEN with an additional N-terminal cysteine for site-directed coupling was fused to AnxA5 (XTEN-AnxA5). The fusion protein could be highly expressed in Escherichia coli and efficiently purified using XTEN conveniently as a purification tag. It was labeled with a thiol-reactive fluorescent dye and via a chelator with a radionuclide. Results: SPECT/CT imaging revealed a blood half-life of about 1 h in mice, markedly longer than the 7-min blood half-life for unmodified AnxA5, which should allow improved imaging of target tissues with low perfusion. In comparison to AnxA5, XTEN-AnxA5 demonstrated a substantially higher accumulation in tumors under chemotherapy in near-infrared fluorescence imaging. Conclusion: The presented method allows the expression and production of high amounts of long-circulating XTEN-AnxA5 without the necessity of PEGylation, thereby simplifying the synthesis while avoiding labeling-induced inactivation of AnxA5 and potential adverse effects of PEG. It is readily applicable to other recombinant protein or peptide-based imaging probes and allows fine-tuning of the desired blood half-life, because longer XTEN variants yield longer blood half-lives.

Gadolinium‐containing magnetic resonance contrast media: investigation on the possible transchelation of Gd3+ to the glycosaminoglycan heparin

Retention of gadolinium (Gd) in biological tissues is considered an important cofactor in the development of nephrogenic systemic fibrosis (NSF). Research on this issue has so far focused on the stability of Gd-based contrast media (GdCM) and a possible release of Gd³⁺ from the complex. No studies have investigated competing chelators that may occur in vivo. We performed proton T(1) -relaxometry in solutions of nine approved GdCM and the macromolecular chelator heparin (250 000 IU per 10 ml) without and with addition of ZnCl₂. For the three linear, nonspecific GdCM complexes, Omniscan®, OptiMARK® and Magnevist®, 2 h of incubation in heparin at 37 °C in the presence of 2.0 mm ZnCl₂ led to an increase in T₁-relaxivity by a factor of 7.7, 5.6 and 5.1, respectively. For the three macrocyclic complexes, Gadovist®, Dotarem® and Prohance®, only a minor increase in T₁-relaxivity by a factor of 1.5, 1.6 and 1.7 was found, respectively. Without addition of ZnCl₂, no difference between the two GdCM groups was observed (factors of 1.4, 1.2, 1.1, 1.3, 1.5 and 1.4, respectively). The increase in T₁-relaxivities observed for linear GdCM complexes may be attributable to partial transchelation with formation of a macromolecular Gd-heparin complex. For comparison, mixing of GdCl₃ and heparin results in a 8.7-fold higher T₁-relaxivity compared with a solution of GdCl₃ in water. Heparin is a glycosaminoglycan (GAG) and as such occurs in the human body as a component of the extracellular matrix. GAGs generally are known to be strong chelators. Gd³⁺ released from chelates of GdCM might be complexed by GAGs in vivo, which would explain their retention in biological tissues. Plasma GAG levels are elevated in end-stage renal disease; hence, our results might contribute to the elucidation of NSF.

Synthetic routes to magnetic nanoparticles for MPI

Abstract Magnetic particle imaging (MPI) is a new imaging technique for visualizing the three-dimensional distribution of superparamagnetic iron oxide nanoparticles with specific properties (MPI tracers). Initial results obtained with MPI using superparamagnetic iron oxide as blood pool markers suggest that the method has great potential for cardiovascular imaging. Conversely, no clinically approved MPI tracers currently exist that could be used to exploit this potential of MPI. This article describes thermal decomposition and coprecipitation, two relevant methods for synthesizing and optimizing superparamagnetic iron oxide nanoparticles for MPI. Furthermore it summarizes the recent literature on MPI tracers and explores what can be learned from structural studies with Resovist® for novel synthesis approaches.

Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist® for improved MRI of MSC with single-cell sensitivity.

Straightforward thiol-mediated protein labelling with DTPA: Synthesis of a highly active 111In-annexin A5-DTPA tracer

BackgroundAnnexin A5 (anxA5) has been found useful for molecular imaging of apoptosis and other biological processes.MethodsHere, we report an optimised two-step synthesis of annexin A5-diethylene triamine pentaacetic acid (DTPA) (anxA5-DTPA) for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a single purification step. The use of a recombinant annexin A5 (cys-anxA5) with a single thiol group allowed regionally specific coupling, without affecting the binding domain of cys-anxA5.ResultsThe metal complexing capacity of anxA5-DTPA was investigated by labelling with 111In3+ and Eu3+. Binding of modified anxA5-DTPA to apoptotic cells was tested in competition experiments with a fluorescent anxA5 derivative (anxA5-FITC) using flow cytometry and compared with that of wildtype anxA5 or non-binding anxA5-DTPA (M1234-anxA5-DTPA). The binding affinity to apoptotic cells of the anxA5-DTPA conjugate does not differ from that of wildtype anxA5.ConclusionsThis two-step synthesis of annexin A5-DTPA resulted in biologically active anxA5-DTPA, which can be labelled with radionuclides for use in SPECT and PET imaging.

Europium doping of superparamagnetic iron oxide nanoparticles enables their detection by fluorescence microscopy and for quantitative analytics

BACKGROUND Pharmacokinetic studies and histological detection of superparamagnetic iron oxide nanoparticles (SPIO) in biomedical research are limited due to a high iron background especially in pathological tissues. OBJECTIVE The suitability of doping the iron oxide cores of SPIO with europium (Eu) was tested for improved histologic detection and for quantitative analysis without changing their properties as probes for magnetic resonance imaging (MRI). A special variant of SPIO, so called very small superparamagnetic iron oxide nanoparticles (VSOP), was used for this approach. METHODS VSOP, stabilized by a citrate coating, were synthesized with and without addition of Eu (Eu-VSOP and VSOP, respectively). MR signal enhancing effects of Eu-VSOP and VSOP were studied in vitro. Cellular uptake of Eu-VSOP and VSOP was examined in RAW264.7 cells. For Eu-VSOP, fluorescence microscopy and spectrophotometry were used. Eu fluorescence was enhanced by means of an antenna system. For VSOP, Prussian blue staining and photometry using the phenanthroline method were applied. Results for both VSOP variants were compared. RESULTS Eu-VSOP and VSOP did not differ with respect to MR signal enhancing effects nor to uptake characteristics in the RAW264.7 cell experiments. Fluorescence microscopy detects Eu-VSOP with higher sensitivity compared to light microscopy using Prussian blue staining. In microscopy as well as in the analytical quantification using fluorescence, detection of Eu-VSOP is not contaminated by Fe background. CONCLUSIONS Doping the VSOP with Eu allows for their improved detection by fluorescence microscopy and quantitative analysis without changing their cellular uptake characteristics or their MR signal enhancing effects and thus would allow for a multimodal approach for studying their pharmacokinetics and biodistribution in experimental research.

Evaluation of Different Magnetic Particle Systems with Respect to Its MPI Performance

The Magnetic Particle Spectroscopy (MPS)-amplitudes were measured on 7 suspensions of magnetite based magnetic particles (MNP) differing in core size and magnetic anisotropy. The distributions of the effective domain sizes, estimated by means of quasistatic M(H) measurements and Magnetorelaxometry (MRX), matches well the core size distribution for the single core MNP-systems estimated by electron microscopy. Two systems, namely Resovist and M4E clearly exhibit a bimodal domain size distribution. It was shown, that the MPS amplitudes strongly increase with increasing domain size up to 21 nm, the mean value of the larger fraction of Resovist. For M4E with a mean size of the larger fraction of 33 nm the measured MPS-amplitudes became much smaller than those of Resovist, in particular for the higher harmonics. That behaviour was attributed to the mean anisotropy energy of these MNPs, estimated by MRX, exceeding that of Resovist by one order of magnitude. The effect of the MNP’s magnetic anisotropy is also supported by comparison of measured MPS-amplitudes with those which were calculated on the base of M(H)-data.

Novel magnetic multicore nanoparticles designed for MPI and other biomedical applications: From synthesis to first in vivo studies

Synthesis of novel magnetic multicore particles (MCP) in the nano range, involves alkaline precipitation of iron(II) chloride in the presence of atmospheric oxygen. This step yields green rust, which is oxidized to obtain magnetic nanoparticles, which probably consist of a magnetite/maghemite mixed-phase. Final growth and annealing at 90°C in the presence of a large excess of carboxymethyl dextran gives MCP very promising magnetic properties for magnetic particle imaging (MPI), an emerging medical imaging modality, and magnetic resonance imaging (MRI). The magnetic nanoparticles are biocompatible and thus potential candidates for future biomedical applications such as cardiovascular imaging, sentinel lymph node mapping in cancer patients, and stem cell tracking. The new MCP that we introduce here have three times higher magnetic particle spectroscopy performance at lower and middle harmonics and five times higher MPS signal strength at higher harmonics compared with Resovist®. In addition, the new MCP have also an improved in vivo MPI performance compared to Resovist®, and we here report the first in vivo MPI investigation of this new generation of magnetic nanoparticles.

Tracer Development for Magnetic Particle Imaging

Magnetic particle imaging (MPI) allows quantitative evaluation of the spatial distribution of superparamagnetic iron oxide (SPIO) nanoparticles in the body. With a spatial resolution similar to magnetic resonance imaging (MRI), but superior temporal resolution, MPI has potential for different diagnostic applications. In addition to technical requirements, preclinical and clinical applications of this novel imaging modality require SPIO tracers optimized for MPI. This article discusses the suitability of Resovist as an MPI tracer and challenges and future prospects of tracer development.