XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic

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

Macrophage uptake switches on OCT contrast of superparamagnetic nanoparticles for imaging of atherosclerotic plaques

Background: 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. Materials and methods: 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 mu L) 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 (TM) OCT imaging system. Results: 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. Conclusion: 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

Multiplex enzyme activity imaging by MALDI-IMS of substrate library conversions

Enzymes are fundamental to biological processes and involved in most pathologies. Here we demonstrate the concept of simultaneously mapping multiple enzyme activities (EA) by applying enzyme substrate libraries to tissue sections and analyzing their conversion by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). To that end, we spray-applied a solution of 20 naturally derived peptides that are known substrates for proteases, kinases, and phosphatases to zinc-fixed paraffin tissue sections of mouse kidneys. After enzyme conversion for 5 to 120 min at 37 °C and matrix application, the tissue sections were imaged by MALDI-IMS. We could image incubation time-dependently 16 of the applied substrates with differing signal intensities and 12 masses of expected products. Utilizing inherent enzyme amplification, EA-IMS can become a powerful tool to locally study multiple, potentially even lowly expressed, enzyme activities, networks, and their pharmaceutical modulation. Differences in the substrate detectability highlight the need for future optimizations

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of In-111-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE(-/-) mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug

Iron(III)‐tCDTA derivatives as MRI contrast agents: Increased T 1 relaxivities at higher magnetic field strength and pH sensing

Purpose: Low molecular weight iron(III) complex-based contrast agents (IBCA) including iron(III) trans-cyclohexane diamine tetraacetic acid [Fe(tCDTA)](-) could serve as alternatives to gadolinium-based contrast agents in MRI. In search for IBCA with enhanced properties, we synthesized derivatives of [Fe(tCDTA)](-) and compared their contrast effects. Methods: Trans-cyclohexane diamine tetraacetic acid (tCDTA) was chemically modified in 2 steps: first the monoanhydride of Trans-cyclohexane diamine tetraacetic acid was generated, and then it was coupled to amines in the second step. After purification, the chelators were analyzed by high-performance liquid chromatography, mass spectrometry, and NMR spectrometry. The chelators were complexed with iron(III), and the relaxivities of the complexes were measured at 0.94, 1.5, 3, and 7 Tesla. Kinetic stabilities of the complexes were analyzed spectrophotometrically and the redox properties by cyclic voltammetry. Results: Using ethylenediamine (en) and trans-1,4-diaminocyclohexane, we generated monomers and dimers of tCDTA: en-tCDTA, en-tCDTA-dimer, trans-1,4-diaminocyclohexane-tCDTA, and trans-1,4-diaminocyclohexane-tCDTA-dimer. The iron(III) complexes of these derivatives had similarly high stabilities as [Fe(tCDTA)](-). The iron(III) complexes of the trans-1,4-diaminocyclohexane derivatives had higher T-1 relaxivities than [Fe(tCDTA)](-) that increased with increasing magnetic field strengths and were highest at 6.8 L.mmol(-1).s(-1) per molecule for the dimer. Remarkably, the relaxivity of [Fe(en-tCDTA)](+) had a threefold increase from neutral pH toward pH6. Conclusion: Four iron(III) complexes with similar stability in comparison to [Fe(tCDTA)](-) were synthesized. The relaxivities of trans-1,4-diaminocyclohexane-tCDTA and trans-1,4-diaminocyclohexane-tCDTA-dimer complexes were in the same range as gadolinium-based contrast agents at 3 Tesla. The [Fe(en-tCDTA)](+) complex is a pH sensor at weakly acidic pH levels, which are typical for various cancer types

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe3+), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that 1H2O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T1 shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T2 linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R2) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future

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

Changes in Liver Mechanical Properties and Water Diffusivity During Normal Pregnancy Are Driven by Cellular Hypertrophy

During pregnancy, the body's hyperestrogenic state alters hepatic metabolism and synthesis. While biochemical changes related to liver function during normal pregnancy are well understood, pregnancy-associated alterations in biophysical properties of the liver remain elusive. In this study, we investigated 26 ex vivo fresh liver specimens harvested from pregnant and non-pregnant rats by diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE) in a 0.5-Tesla compact magnetic resonance imaging (MRI) scanner. Water diffusivity and viscoelastic parameters were compared with histological data and blood markers. We found livers from pregnant rats to have (i) significantly enlarged hepatocytes (26 ± 15%, p < 0.001), (ii) increased liver stiffness (12 ± 15%, p = 0.012), (iii) decreased viscosity (-23 ± 14%, p < 0.001), and (iv) increased water diffusivity (12 ± 11%, p < 0.001). In conclusion, increased stiffness and reduced viscosity of the liver during pregnancy are mainly attributable to hepatocyte enlargement. Hypertrophy of liver cells imposes fewer restrictions on intracellular water mobility, resulting in a higher hepatic water diffusion coefficient. Collectively, MRE and DWI have the potential to inform on structural liver changes associated with pregnancy in a clinical context

Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect

Anoctamins are a family of Ca$^{2+}$-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca$^{2+}$ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patchclamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca$^{2+}$-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca$^{2+}$-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease

Construction of the recombinant cytostatic/cytotoxic protein XTEN-Killin.

<p>Schematic representation of the DNA/protein sequence of XTEN-Killin (A), the procedure for production (B), and a summary of its intended functions (C).</p