Magnetic response of gelatin ferrogels across the sol-gel transition: the influence of high energy crosslinking on thermal stability.

As emerging responsive materials, ferrogels have demonstrated significant potential for applications in areas of engineering to regenerative medicine. Promising techniques to study the behavior of magnetic nanoparticles (MNPs) in such matrices include magnetic particle spectroscopy (MPS) and magnetorelaxometry (MRX). This work investigated the magnetic response of gelatin-based ferrogels with increasing temperatures, before and after high energy crosslinking. The particle response was characterized by the nonlinear magnetization using MPS and quasistatic magnetization measurements as well as MRX to discriminate between Néel and Brownian relaxation mechanisms. The effective magnetic response of MNPs in gelatin was suppressed, indicating that the magnetization of the ferrogels was strongly influenced by competing dipole-dipole interactions. Significant changes in the magnetic behavior were observed across the gelatin sol-gel transition, as influenced by the matrix viscosity. These relaxation processes were modeled by Fourier transformation of the Langevin function, combined with a Debye term for the nonlinear magnetic response, for single core MNPs embedded in matrices of changing viscosities. Using high energy electron irradiation as a crosslinking method, modified ferrogels exhibited thermal stability on a range of timescales. However, MRX relaxation times revealed a slight softening around the gelatin sol-gel transition felt by the smallest particles, demonstrating a high sensitivity to observe local changes in the viscoelasticity. Overall, MPS and MRX functioned as non-contact methods to observe changes in the nanorheology around the native sol-gel transition and in crosslinked ferrogels, as well as provided an understanding of how MNPs were integrated into and influenced by the surrounding matrix.

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.

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.

Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Determining the resolution of a tracer for magnetic particle imaging by means of magnetic particle spectroscopy.

Magnetic particle imaging (MPI) is an imaging modality to quantitatively determine the three-dimensional distribution of magnetic nanoparticles (MNPs) administered as a tracer into a biological system. Magnetic particle spectroscopy (MPS) is the zero-dimensional MPI counterpart without spatial coding but with much higher sensitivity. Generally, MPS is employed to qualitatively evaluate the MPI capability of tracer systems from the measured specific harmonic spectra. Here, we investigated the correlation of three characteristic MPS parameters with the achievable MPI resolution from a recently introduced procedure based on a two-voxel-analysis of data taken from the system function acquisition that is mandatory in Lissajous scanning MPI. We evaluated nine different tracer systems and determined their MPI capability and resolution from MPS measurements and compared the results with MPI phantom measurements.

Tailored Magnetic Multicore Nanoparticles for Use as Blood Pool MPI Tracers.

For the preclinical development of magnetic particle imaging (MPI) in general, and the exploration of possible new clinical applications of MPI in particular, tailored MPI tracers with surface properties optimized for the intended use are needed. Here we present the synthesis of magnetic multicore particles (MCPs) modified with polyethylene glycol (PEG) for use as blood pool MPI tracers. To achieve the stealth effect the carboxylic groups of the parent MCP were activated and coupled with pegylated amines (mPEG-amines) with different PEG-chain lengths from 2 to 20 kDa. The resulting MCP-PEG variants with PEG-chain lengths of 10 kDa (MCP-PEG10K after one pegylation step and MCP-PEG10K2 after a second pegylation step) formed stable dispersions and showed strong evidence of a successful reaction of MCP and MCP-PEG10K with mPEG-amine with 10 kDa, while maintaining their magnetic properties. In rats, the mean blood half-lives, surprisingly, were 2 and 62 min, respectively, and therefore, for MCP-PEG10K2, dramatically extended compared to the parent MCP, presumably due to the higher PEG density on the particle surface, which may lead to a lower phagocytosis rate. Because of their significantly extended blood half-life, MCP-PEG10K2 are very promising as blood pool tracers for future in vivo cardiovascular MPI.

In vivo magnetic particle imaging: angiography of inferior vena cava and aorta in rats using newly developed multicore particles.

Magnetic Particle Imaging (MPI) is a new imaging modality, which maps the distribution of magnetic nanoparticles (MNP) in 3D with high temporal resolution. It thus may be suited for cardiovascular imaging. Its sensitivity and spatial resolution critically depend on the magnetic properties of MNP. Therefore, we used novel multicore nanoparticles (MCP 3) for in-vivo MPI in rats and analyzed dose requirements, sensitivity and detail resolution. 8 rats were examined using a preclinical MPI scanner (Bruker Biospin GmbH, Germany) equipped with a separate receive coil. MCP 3 and Resovist were administered intravenously (i.v.) into the rats' tail veins at doses of 0.1, 0.05 and 0.025 mmol Fe/kg followed by serial MPI acquisition with a temporal resolution of 46 volumes per second. Based on a qualitative visual scoring system MCP 3-MPI images showed a significantly (P ≤ 0.05) higher image quality than Resovist-MPI images. Morphological features such as vessel lumen diameters (DL) of the inferior vena cava (IVC) and abdominal aorta (AA) could be assessed along a 2-cm segment in mesenteric area only after administration of MCP 3 at dosages of 0.1, 0.05 mmol Fe/kg. The mean DL ± SD estimated was 2.7 ± 0.6 mm for IVC and 2.4 ± 0.7 mm for AA. Evaluation of DL of the IVC and AA was not possible in Resovist-MPI images. Our results show, that MCP 3 provide better image quality at a lower dosage than Resovist. MCP 3-MPI with a clinically acceptable dose of 0.05 mmol Fe/kg increased the visibility of vessel lumens compared to Resovist-based MPI towards possible detection of vascular abnormalities such as stenosis or aneurysms, in vivo.

MPI Phantom Study with A High-Performing Multicore Tracer Made by Coprecipitation.

Magnetic particle imaging (MPI) is a new imaging technique that detects the spatial distribution of magnetic nanoparticles (MNP) with the option of high temporal resolution. MPI relies on particular MNP as tracers with tailored characteristics for improvement of sensitivity and image resolution. For this reason, we developed optimized multicore particles (MCP 3) made by coprecipitation via synthesis of green rust and subsequent oxidation to iron oxide cores consisting of a magnetite/maghemite mixed phase. MCP 3 shows high saturation magnetization close to that of bulk maghemite and provides excellent magnetic particle spectroscopy properties which are superior to Resovist® and any other up to now published MPI tracers made by coprecipitation. To evaluate the MPI characteristics of MCP 3 two kinds of tube phantoms were prepared and investigated to assess sensitivity, spatial resolution, artifact severity, and selectivity. Resovist® was used as standard of comparison. For image reconstruction, the regularization factor was optimized, and the resulting images were investigated in terms of quantifying of volumes and iron content. Our results demonstrate the superiority of MCP 3 over Resovist® for all investigated MPI characteristics and suggest that MCP 3 is promising for future experimental in vivo studies.

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.

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.

LA-ICP-MS Allows Quantitative Microscopy of Europium-Doped Iron Oxide Nanoparticles and is a Possible Alternative to Ambiguous Prussian Blue Iron Staining.

The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections. For synthesis of VSOP, a fraction of Fe3+ (5 wt%) was replaced by Eu3+, resulting in particles with 0.66 mol% europium relative to iron (Eu-VSOP) but with otherwise similar properties as VSOP. Eu-VSOP or VSOP was intravenously injected into ApoE-/- mice on Western cholesterol diet and accumulated in atherosclerotic plaques of these animals. Prussian blue staining was positive for ApoE-/- mice with particle injection but also for controls. LA-ICP-MS microscopy resulted in sensitive and specific detection of the europium of Eu-VSOP in liver and atherosclerotic plaques. Furthermore, calibration with Eu-VSOP allowed calculation of iron and particle concentrations in tissue sections. The combination of europium-doped iron oxide particles and LA-ICP-MS microscopy provides a new tool for specific and quantitative analysis of particle distribution at the tissue level and allows correlation with other elements such as endogenous iron.

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.

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

UNLABELLED: 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.

Synthetic routes to magnetic nanoparticles for MPI.

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.

Gadolinium-containing magnetic resonance contrast media: investigation on the possible transchelation of Gd³âº 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 Gd³âº 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. Gd³âº 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.

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

BACKGROUND: Annexin A5 (anxA5) has been found useful for molecular imaging of apoptosis and other biological processes. METHODS: Here, 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. RESULTS: The 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. CONCLUSIONS: This 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.