Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Pulsed Optically Pumped Magnetometers: Addressing Dead Time and Bandwidth for the Unshielded Magnetorelaxometry of Magnetic Nanoparticles.

Magnetic nanoparticles (MNP) offer a large variety of promising applications in medicine thanks to their exciting physical properties, e.g., magnetic hyperthermia and magnetic drug targeting. For these applications, it is crucial to quantify the amount of MNP in their specific binding state. This information can be obtained by means of magnetorelaxometry (MRX), where the relaxation of previously aligned magnetic moments of MNP is measured. Current MRX with optically pumped magnetometers (OPM) is limited by OPM recovery time after the shut-off of the external magnetic field for MNP alignment, therewith preventing the detection of fast relaxing MNP. We present a setup for OPM-MRX measurements using a commercially available pulsed free-precession OPM, where the use of a high power pulsed pump laser in the sensor enables a system recovery time in the microsecond range. Besides, magnetometer raw data processing techniques for Larmor frequency analysis are proposed and compared in this paper. Due to the high bandwidth (≥100 kHz) and high dynamic range of our OPM, a software gradiometer in a compact enclosure allows for unshielded MRX measurements in a laboratory environment. When operated in the MRX mode with non-optimal pumping performance, the OPM shows an unshielded gradiometric noise floor of about 600 fT/cm/Hz for a 2.3 cm baseline. The noise floor is flat up to 1 kHz and increases then linearly with the frequency. We demonstrate that quantitative unshielded MRX measurements of fast relaxing, water suspended MNP is possible with the novel OPM-MRX concept, confirmed by the accurately derived iron amount ratios of MNP samples. The detection limit of the current setup is about 1.37 µg of iron for a liquid BNF-MNP-sample (Bionized NanoFerrite) with a volume of 100 µL.

2D Quantitative Imaging of Magnetic Nanoparticles by an AC Biosusceptometry Based Scanning Approach and Inverse Problem.

The use of magnetic nanoparticles (MNPs) in biomedical applications requires the quantitative knowledge of their quantitative distribution within the body. AC Biosusceptometry (ACB) is a biomagnetic technique recently employed to detect MNPs in vivo by measuring the MNPs response when exposed to an alternate magnetic field. The ACB technique presents some interesting characteristics: non-invasiveness, low operational cost, high portability, and no need for magnetic shielding. ACB conventional methods until now provided only qualitative information about the MNPs' mapping in small animals. We present a theoretical model and experimentally demonstrate the feasibility of ACB reconstructing 2D quantitative images of MNPs' distributions. We employed an ACB single-channel scanning approach, measuring at 361 sensor positions, to reconstruct MNPs' spatial distributions. For this, we established a discrete forward problem and solved the ACB system's inverse problem. Thus, we were able to determine the positions and quantities of MNPs in a field of view of 5×5×1 cm3 with good precision and accuracy. The results show the ACB system's capabilities to reconstruct the quantitative spatial distribution of MNPs with a spatial resolution better than 1 cm, and a sensitivity of 1.17 mg of MNPs fixed in gypsum. These results show the system's potential for biomedical application of MNPs in several studies, for example, electrochemical-functionalized MNPs for cancer cell targeting, quantitative sensing, and possibly in vivo imaging.

Quantitative 2D Magnetorelaxometry Imaging of Magnetic Nanoparticles using Optically Pumped Magnetometers.

For biomagnetical applications exploiting physical properties of magnetic nanoparticles (MNP), e.g., magnetic hyperthermia, knowledge about the quantitative spatial MNP distribution is crucial, which can be extracted by magnetorelaxometry (MRX) imaging. In this paper, we present quantification, quantitative 1D reconstruction, and quantitative 2D imaging of MNP by exploiting optically pumped magnetometers for MRX. While highlighting the potential of commercially available optically pumped magnetometers (OPM) for MRXI, we discuss current limitations of the used OPM. We show, that with our OPM setup, MNP can be precisely quantified with iron amounts down to ≈ 6 g , which can be improved easily. With a 1D-reconstruction setup, point-like and complex MNP phantoms can be reconstructed quantitatively with high precision and accuracy. We show that with our developed 2D MRX imaging setup, which measures 12 c m by 8 c m , point-like MNP distributions with clinically relevant iron concentrations can be reconstructed precisely and accurately. Our 2D setup has the potential to be easily extended to a tomography styled (and thus slice-selective) 3D scanner, by adding a mechanical axis to the phantom.

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.

Human-sized quantitative imaging of magnetic nanoparticles with nonlinear magnetorelaxometry.

Objective.Magnetorelaxomety imaging (MRXI) is a noninvasive imaging technique for quantitative detection of magnetic nanoparticles (MNPs). The qualitative and quantitative knowledge of the MNP distribution inside the body is a prerequisite for a number of arising biomedical applications, such as magnetic drug targeting and magnetic hyperthermia therapy. It was shown throughout numerous studies that MRXI is able to successfully localize and quantify MNP ensembles in volumes up to the size of a human head. However, deeper regions that lie far from the excitation coils and the magnetic sensors are harder to reconstruct due to the weaker signals from the MNPs in these areas. On the one hand, stronger magnetic fields need to be applied to produce measurable signals from such MNP distributions to further upscale MRXI, on the other hand, this invalidates the assumption of a linear relation between applied magnetic field and particle magnetization in the current MRXI forward model which is required for the imaging procedure.Approach.We tackle this problem by introducing a nonlinear MRXI forward model that is also valid for strong magnetic excitation fields.Main results.We demonstrate in our experimental feasibility study that scaling up the imaging region to the size of a human torso using nonlinear MRXI is possible. Despite the extreme simplicity of the imaging setup applied in this study, an immobilized MNP sample with 6.3 cm3and 12 mg Fe could be localized and quantified with an acceptable quality.Significance.A well-engineered MRXI setup could provide much better imaging qualities in shorter data acquisition times, making nonlinear MRXI a viable option for the supervision of MNP related therapies in all regions of the human body, specifically magnetic hyperthermia.

Experimental demonstration of improved magnetorelaxometry imaging performance using optimized coil configurations.

BACKGROUND: Magnetorelaxometry imaging is an experimental imaging technique capable of reconstructing magnetic nanoparticle distributions inside a volume noninvasively and with high specificity. Thus, magnetorelaxometry imaging is a promising candidate for monitoring a number of therapeutical approaches that employ magnetic nanoparticles, such as magnetic drug targeting and magnetic hyperthermia, to guarantee their safety and efficacy. Prior to a potential clinical application of this imaging modality, it is necessary to optimize magnetorelaxometry imaging systems to produce reliable imaging results and to maximize the reconstruction accuracy of the magnetic nanoparticle distributions. Multiple optimization approaches were already applied throughout a number of simulation studies, all of which yielded increased imaging qualities compared to intuitively designed measurement setups. PURPOSE: None of these simulative approaches was conducted in practice such that it still remains unclear if the theoretical results are achievable in an experimental setting. In this study, we demonstrate the technical feasibility and the increased reconstruction accuracy of optimized coil configurations in two distinct magnetorelaxometry setups. METHODS: The electromagnetic coil positions and radii of a cuboidal as well as a cylindrical magnetorelaxometry imaging setup are optimized by minimizing the system matrix condition numbers of their corresponding linear forward models. The optimized coil configurations are manufactured alongside with two regular coil grids. Magnetorelaxometry measurements of three cuboidal and four cylindrical magnetic nanoparticle phantoms are conducted, and the resulting reconstruction qualities of the optimized and the regular coil configurations are compared. RESULTS: The computed condition numbers of the optimized coil configurations are approximately one order of magnitude lower compared to the regular coil grids. The reconstruction results show that for both setups, every phantom is recovered more accurately by the optimized coil configurations compared to the regular coil grids. Additionally, the optimized coil configurations yield better signal qualities. CONCLUSIONS: The presented experimental study provides a proof of the practicality and the efficacy of optimizing magnetorelaxometry imaging systems with respect to the condition numbers of their system matrices, previously only demonstrated in simulations. From the promising results of our study, we infer that the minimization of the system matrix condition number will also enable the practical optimization of other design parameters of magnetorelaxometry imaging setups (e.g., sensor configuration, coil currents, etc.) in order to improve the achievable reconstruction qualities even further, eventually paving the way towards clinical application of this imaging modality.

Developing magnetorelaxometry imaging for human applications.

Objective.Magnetic nanoparticles (MNPs) are a promising tool in biomedical applications such as cancer therapy and diagnosis, where localization and quantification of MNP distributions are often mandatory. This can be obtained by magnetorelaxometry imaging (MRXI).Approach.In this work, the capability of MRXI for quantitative imaging of MNP inside larger volumes such as a human head is investigated. We developed a human head phantom simulating a glioblastoma multiforme (GBM) tumor containing MNP for magnetic hyperthermia treatment. The sensitivity of our MRXI setup for detection of MNP concentrations in the range of 3-19 mg cm-3was studied.Main result.The results show the high capability of MRXI to detect MNPs in a human head sized volume. Superficial sources with a concentration larger than 12 mg cm-3could be reconstructed with a resulotion of about 1 cm-3.Significance.The reconstruction of the MNP distribution, mimicking a GBM tumor of 7 cm3volume with clinically relevant iron concentration, demonstrates thein vivofeasibility of MRXI in humans.

Quantitative imaging of magnetic nanoparticles in an unshielded environment using a large AC susceptibility array.

BACKGROUND: Non-invasive magnetic imaging techniques are necessary to assist magnetic nanoparticles in biomedical applications, mainly detecting their distribution inside the body. In Alternating Current Biosusceptometry (ACB), the magnetic nanoparticle's magnetization response under an oscillating magnetic field, which is applied through an excitation coil, is detected with a balanced detection coil system. RESULTS: We built a Multi-Channel ACB system (MC-ACB) containing nineteen pick-up coils and obtained 2D quantitative images of magnetic nanoparticle distributions by solving an inverse problem. We reconstructed the magnetic nanoparticles spatial distributions in a field of view of 14 × 14 cm2 with a spatial resolution of 2.0 cm and sensitivity in the milligram scale. A correlation coefficient between quantitative reconstructed and nominal magnetic nanoparticle distributions above 0.6 was found for all measurements. CONCLUSION: Besides other interesting features such as sufficient large field of view dimension for mice and rat studies, portability, and the ability to assess the quantitative magnetic nanoparticles distributions in real-time, the MC-ACB system is a promising tool for quantitative imaging of magnetic nanoparticles distributions in real-time, offering an affordable setup for easy access in clinical or laboratory environments.

Noninvasive monitoring of blood flow using a single magnetic microsphere.

Noninvasive medical imaging of blood flow relies on mapping the transit of a contrast medium bolus injected intravenously. This has the draw-back that the front of the bolus widens until the tissue of interest is reached and quantitative flow parameters are not easy to obtain. Here, we introduce high resolution (millimeter/millisecond) 3D magnetic tracking of a single microsphere locally probing the flow while passing through a vessel. With this, we successfully localize and evaluate diameter constrictions in an arteria phantom after a single passage of a microsphere. We further demonstrate the potential for clinical application by tracking a microsphere smaller than a red blood cell.

Magnetorelaxometry procedures for quantitative imaging and characterization of magnetic nanoparticles in biomedical applications.

BACKGROUND: Quantitative knowledge about the spatial distribution and local environment of magnetic nanoparticles (MNPs) inside an organism is essential for guidance and improvement of biomedical applications such as magnetic hyperthermia and magnetic drug targeting. Magnetorelaxometry (MRX) provides such quantitative information by detecting the magnetic response of MNPs following a fast change in the applied magnetic field. METHODS: In this article, we review our MRX based procedures that enable both the characterization and the quantitative imaging of MNPs in a biomedical environment. RESULTS: MRX characterization supported the selection of an MNP system with colloidal stability and suitable cellular MNP uptake. Spatially resolved MRX, a procedure employing multi-channel MRX measurements allowed for in-vivo monitoring of the MNP distribution in a pre-clinical carcinoma animal model. Extending spatially resolved MRX by consecutive magnetization of distinct parts of the sample led to a demonstration of MRX tomography. With this tomography, we reconstructed the three dimensional MNP distribution inside animal sized phantoms with a sensitivity of milligrams of MNPs per cm3. In addition, the targeting efficiency of MNPs in whole blood was assessed using a flow phantom and MRX quantification. CONCLUSION: These MRX based measurement and analysis procedures have substantially supported the development of MNP based biomedical applications.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

Noninvasive quantification of magnetic nanoparticles by means of magnetorelaxometry.

Meaningful applications of magnetic nanoparticles (MNP) in biomedicine require the ability to quantify MNP in a variety of environments and biological materials (cells, tissue, and organs). Here we describe a method for the integral, noninvasive quantification of MNP. Our method is based on magnetorelaxometry (MRX), it relies on the detection of their magnetic stray fields which permeate practically unaltered virtually any biological material. MRX provides a specific signal which is not affected by dia- and paramagnetic signals from the biological matrix. We describe how to conduct the MRX experiments and use MNP-assisted gene transfection as the example. The quantification is based on the comparison of the MRX signal of a sample and an appropriate reference sample.