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Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli-a concept that could lead to efficient production of highly multifunctional drugs in the future.

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.

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.

Background Intrinsic iron in biological tissues frequently precludes unambiguous the identification of iron oxide nanoparticles when iron-based detection methods are used. Here we report the full methodology for synthesizing very small iron oxide nanoparticles (VSOP) doped with europium (Eu) in their iron oxide core (Eu-VSOP) and their unambiguous qualitative and quantitative detection by fluorescence. Methods and results The resulting Eu-VSOP contained 0.7 to 2.7% Eu relative to iron, which was sufficient for fluorescent detection while not altering other important particle parameters such as size, surface charge, or relaxivity. A customized enhancer solution with high buffer capacity and nearly neutral pH was developed to provide an antenna system that allowed fluorescent detection of Eu-VSOP in cells and histologic tissue slices as well as in solutions even under acidic conditions as frequently obtained from dissolved organic material. This enhancer solution allowed detection of Eu-VSOP using a standard fluorescence spectrophotometer and a fluorescence microscope equipped with a custom filter set with an excitation wavelength (λ ex ) of 338 nm and an emission wavelength (λ em ) of 616 nm. Conclusion The fluorescent detection of Eu-doped very small iron oxide nanoparticles (Eu-VSOP) provides a straightforward tool to unambiguously characterize VSOP biodistribution and toxicology at tissue, and cellular levels, providing a sensitive analytical tool to detect Eu-doped IONP in dissolved organ tissue and biological fluids with fluorescence instruments.

Microscopic structural alterations of liver tissue induced by freeze-thaw cycles give rise to palpable property changes. However, the underlying damage to tissue architecture is difficult to quantify histologically, and published data on macroscopic changes in biophysical properties are sparse. To better understand the influence of hepatic cells and stroma on global biophysical parameters, we studied rat liver specimens freshly taken (within 30 min after death) and treated by freeze-thaw cycles overnight at either −20 °C or –80 °C using diffusion-weighted imaging (DWI) and multifrequency magnetic resonance elastography (MRE) performed at 0.5 T in a tabletop MRE scanner. Tissue structure was analyzed histologically and rheologic data were analyzed using fractional order derivatives conceptualized by a called spring-pot component that interpolates between pure elastic and viscous responses. Overnight freezing and thawing induced membrane disruptions and cell detachment in the space of Disse, resulting in a markedly lower shear modulus μ and apparent diffusion coefficient (ADC) (μ[−20 °C] = 1.23 ± 0.73 kPa, μ[−80 °C] = 0.66 ± 0.75 kPa; ADC[–20 °C] = 0.649 ± 0.028 μm2/s, ADC[−80 °C] = 0.626 ± 0.025 μm2/s) compared to normal tissue (μ = 9.92 ± 3.30 kPa, ADC = 0.770 ± 0.023 μm2/s, all p < 0.001). Furthermore, we analyzed the springpot-powerlaw coefficient and observed a reduction in −20 °C specimens (0.22 ± 0.14) compared to native tissue (0.40 ± 0.10, p = 0.033) and −80 °C specimens (0.54 ± 0.22, p = 0.002), that correlated with histological observations of sinusoidal dilation and collagen distortion within the space of Disse. Overall, the results suggest that shear modulus and water diffusion in liver tissue markedly decrease due to cell membrane degradation and cell detachment while viscosity-related properties appear to be more sensitive to distorted stromal and microvascular architecture.

Optical coherence tomography (OCT) is an intravascular, high-resolution imaging technique that is used to characterize atherosclerotic plaques. However, the identification of macrophages as important markers of inflammation and plaque vulnerability remains difficult. Here, we investigate whether the uptake of very small iron oxide particles (VSOP) in macrophages, that cluster in phagolysosomes and allow high-quality magnetic resonance imaging (MRI) of atherosclerotic plaques, and uptake of ferumoxytol nanoparticles enhance detection of macrophages by OCT. RAW 264.7 macrophage cells were incubated with VSOP (1 and 2 mM Fe) that have been clinically tested and ferumoxytol (8.9 mM Fe) that is approved for iron deficiency treatment and currently investigated as an MRI contrast agent. The light scattering of control macrophages, nanoparticle-labeled macrophages (2,000,000 in 500 µL) and nanoparticle suspensions was measured in synchronous wavelength scan mode using a fluorescence spectrophotometer. For OCT analyses, pellets of 8,000,000 non-labeled, VSOP-labeled and ferumoxytol-labeled RAW 264.7 macrophages were imaged and analyzed on an OPTIS™ OCT imaging system. Incubation with 1 and 2 mM VSOP resulted in uptake of 7.1±1.5 and 12±1.5 pg Fe per cell, which increased the backscattering of the macrophages in spectrophotometry 2.5- and 3.6-fold, whereas incubation with 8.9 mM Fe ferumoxytol resulted in uptake of 6.6±2 pg Fe per cell, which increased the backscattering 1.5-fold at 700 nm. In contrast, backscattering of non-clustered nanoparticles in suspension was negligible. Accordingly, OCT imaging could visualize significantly increased backscattering and signal attenuation of nanoparticle-labeled macrophages in comparison with controls. We conclude that VSOP and, to a lesser extent, ferumoxytol increase light scattering and attenuation when taken up by macrophages and can serve as a multimodal imaging probe for MRI and OCT to improve macrophage detection in atherosclerotic plaques by OCT in the future.

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.