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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 111In-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.
In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling.
Atherosklerotische Läsionen können durch Magnetresonanztomographie (MRT) unter Verwendung von sehr kleinen Eisenoxidnanopartikeln (VSOP) sichtbar gemacht werden. VSOP akkumulieren in atherosklerotischen Plaques und dienen somit als Atherosklerosesonde.
Das Ziel dieses Projektes war die Abbildung der Verteilung von Europium-dotierten VSOP (Eu-VSOP) durch Laserablation ICP-MS in histologischen Dünnschnitten der Aortenwurzelregion des ApoE-Knockout-Mausmodells, das reich an atherosklerotischen Plaques ist. Darüber hinaus wurde untersucht, ob die VSOP-Akkumulation in den Plaques mit anderen Biomarkern der Entzündung wie Makrophagen und verändertem Endothel korreliert. Somit wäre eine Beurteilung, ob es sich hierbei um instabile bzw. vulnerable Plaque-Regionen handelt möglich. Zu diesem Zweck wurden Antikörper mit verschiedenen Lanthaniden markiert und mit der Eu-VSOP-Verteilung unter Verwendung von LA-ICP-MS in einem Multiplex-Messmodus korreliert.
Eine mögliche Korrelation von reaktiven Stickstoffspezies (RNS) mit endogenem Eisen oder Eu-VSOP kann ebenfalls durch LA-ICP-MS nachgewiesen werden. Zu diesem Zweck wurden RNS-spezifische Antikörper auch mit Lanthaniden markiert.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA-ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe.
The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
A possible correlation of reactive nitrogen species (RNS) with endogenous iron or Eu-VSOP can also be detected by LA-ICP-MS. For this purpose, RNS-specific antibodies were also labeled with lanthanides.
Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat- denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the Problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG- supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250mW and 1500mW was utilized. The Albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and Primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1mmaround the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe.
The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
A possible correlation of reactive oxygen species (ROS) with endogenous iron or Eu-VSOP can also be detected by LA-ICP-MS. For this purpose, ROS-specific antibodies were also labeled with lanthanides.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA-ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe. The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.
Laser ablation with inductively coupled plasma is still more used in life science as biology and biomedicine and the utilization of metals and proteins determination simultaneously is also growing up. We have developed a new strategy of labeling of antibody (it can specific binds to proteins) by nanoparticles and quantum dots which is composed of thousands of atoms and thus increases the sensitivity enormously and of course decreases the Limit of detection, compare to lanthanoids labeling. The ability of successfully tagged antibodies bound to Antigen (protein) was proved by dot blot on membrane imaged by LA-ICP-MS.