Magnetically responsive composites: electron beam assisted magnetic nanoparticle arrest in gelatin hydrogels for bioactuation.

As emerging responsive materials, ferrogels have become highly attractive for biomedical and technical applications in terms of soft actuation, tissue engineering or controlled drug release. In the present study, bioderived ferrogels were fabricated and successfully deformed within moderate, heterogeneous magnetic fields. Synthesis was realized by arresting iron oxide nanoparticles in porcine gelatin by introduction of covalent crosslinks via treatment with energetic electrons for mesh refinement. This approach also allows for tuning thermal and mechanical stability of the gelatin matrix. Operating the bioferrogel in compression, magnetic forces on the nanoparticles are counterbalanced by the stiffness of the hydrogel matrix that is governed by a shift in thermodynamic equilibrium of swelling, as derived in the framework of osmosis. As gelatin and iron oxide nanoparticles are established as biocompatible constituents, these findings promise potential for in vivo use as contactless mechanical transducers.

Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for Targeted Fibrin Clot Dissolution.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention in many biomedical fields and are used in preclinical/experimental drug delivery, hyperthermia and medical imaging. In this study, biocompatible magnetite drug carriers, stabilized by a dextran shell, were developed to carry tissue plasminogen activator (tPA) for targeted thrombolysis under an external magnetic field. Different concentrations of active tPA were immobilized on carboxylated nanoparticles through carbodiimide-mediated amide bond formation. Evidence for successful functionalization of SPIONs with carboxyl groups was shown by Fourier transform infrared spectroscopy. Surface properties after tPA immobilization were altered as demonstrated by dynamic light scattering and ζ potential measurements. The enzyme activity of SPION-bound tPA was determined by digestion of fibrin-containing agarose gels and corresponded to about 74% of free tPA activity. Particles were stored for three weeks before a slight decrease in activity was observed. tPA-loaded SPIONs were navigated into thrombus-mimicking gels by external magnets, proving effective drug targeting without losing the protein. Furthermore, all synthesized types of nanoparticles were well tolerated in cell culture experiments with human umbilical vein endothelial cells, indicating their potential utility for future therapeutic applications in thromboembolic diseases.

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.

Nanomagnetic activation as a way to control the efficacy of nucleic acid delivery.

PURPOSE: To explore the potential of magnetofection in delivering pDNA to primary mouse embryonic fibroblasts (PMEFs) and porcine fetal fibroblasts (PFFs) and investigate an effect of magnetic cell labeling on transfection efficacy. METHODS: The formulation and a dose of the magnetic vector were optimized. The efficacy of the procedure was quantified by vector internalization, transgene expression and cell iron loading upon specific labeling with Ab-conjugated magnetic beads or non-specific labeling with MNPs. RESULTS: Up to sixty percent of PMEF and PFF cells were transfected at low pDNA doses of 4-16 pg pDNA/cell. Specific labeling of the PMEFs with MNPs, resulted in a 3- and 2-fold increase in pDNA internalization upon magnetofection and lipofection, respectively, that yielded a 2-4-fold increase in percent of transgene-expressing cells. Non-specific cell labeling had no effect on the efficacy of the reporter expression, despite the acquisition of similar magnetic moments per cell. In PFFs, specific magnetic labeling of the cell surface receptors inhibited internalization and transfection efficacy. CONCLUSIONS: Magnetic labeling of cell-surface receptors combined with the application of an inhomogenous magnetic field (nanomagnetic activation) can affect the receptor-mediated internalization of delivery vectors and be used to control nucleic acid delivery to cells.

Temperature dependent magnetorelaxometry of magnetic nanoparticle ensembles.

Magnetorelaxometry imaging (MRXI) is a non-invasive, quantitative imaging technique for magnetic nanoparticles (MNPs). The image resolution of this technique significantly depends on the relaxation amplitude (ΔB). For this work, we measured the room temperature (299 K) relaxation signals of eight commercial MNP sample systems with different magnetic properties, in both fluid and immobilized states, in order to select the most suitable sample for a particular MRXI setting. Additionally, the effect of elevated temperatures (up to hyperthermia temperature, 335 K) on the relaxation signals of four different MNP systems (Synomag, Perimag, BNF and Nanomag) in both states were investigated. The ΔBvalues of fluid samples significantly decreased with increasing temperature, and the behaviour for immobilized samples depended on their blocking temperature (TB). For samples withTB< 299 K, ΔBalso decreased with increasing temperature. Whereas for samples withTB> 299 K, the opposite behaviour was observed. These results are beneficial for improving the image resolution in MRXI and show, among the investigated systems, and for our setup, Synomag is the best candidate for futurein vitroandin vivostudies. This is due to its consistently high ΔBbetween 299 and 335 K in both states. Our findings demonstrate the feasibility of temperature imaging by MRXI.

Combined use of magnetic microbeads for endothelial cell isolation and enhanced cell engraftment in myocardial repair.

Background: The regenerative potential of the heart after injury is limited. Therefore, cell replacement strategies have been developed. However, the engraftment of transplanted cells in the myocardium is very inefficient. In addition, the use of heterogeneous cell populations precludes the reproducibility of the outcome. Methods: To address both issues, in this proof of principle study, we applied magnetic microbeads for combined isolation of eGFP+ embryonic cardiac endothelial cells (CECs) by antigen-specific magnet-associated cell sorting (MACS) and improved engraftment of these cells in myocardial infarction by magnetic fields. Results: MACS provided CECs of high purity decorated with magnetic microbeads. In vitro experiments revealed that the angiogenic potential of microbead-labeled CECs was preserved and the magnetic moment of the cells was strong enough for site-specific positioning by a magnetic field. After myocardial infarction in mice, intramyocardial CEC injection in the presence of a magnet resulted in a strong improvement of cell engraftment and eGFP+ vascular network formation in the hearts. Hemodynamic and morphometric analysis demonstrated augmented heart function and reduced infarct size only when a magnetic field was applied. Conclusion: Thus, the combined use of magnetic microbeads for cell isolation and enhanced cell engraftment in the presence of a magnetic field is a powerful approach to improve cell transplantation strategies in the heart.

Quantitative measurement of the magnetic moment of individual magnetic nanoparticles by magnetic force microscopy.

The quantitative measurement of the magnetization of individual magnetic nanoparticles (MNPs) using magnetic force microscopy (MFM) is described. Quantitative measurement is realized by calibration of the MFM signal using an MNP reference sample with traceably determined magnetization. A resolution of the magnetic moment of the order of 10(-18) A m(2) under ambient conditions is demonstrated, which is presently limited by the tip's magnetic moment and the noise level of the instrument. The calibration scheme can be applied to practically any magnetic force microscope and tip, thus allowing a wide range of future applications, for example in nanomagnetism and biotechnology.

Magnetorelaxometry assisting biomedical applications of magnetic nanoparticles.

Due to their biocompatibility and small size, iron oxide magnetic nanoparticles (MNP) can be guided to virtually every biological environment. MNP are susceptible to external magnetic fields and can thus be used for transport of drugs and genes, for heat generation in magnetic hyperthermia or for contrast enhancement in magnetic resonance imaging of biological tissue. At the same time, their magnetic properties allow one to develop sensitive and specific measurement methods to non-invasively detect MNP, to quantify MNP distribution in tissue and to determine their binding state. In this article, we review the application of magnetorelaxometry (MRX) for MNP detection. The underlying physical properties of MNP responsible for the generation of the MRX signal with its characteristic parameters of relaxation amplitude and relaxation time are described. Existing single and multi-channel MRX devices are reviewed. Finally, we thoroughly describe some applications of MRX to cellular MNP quantification, MNP organ distribution and MNP-based binding assays. Providing specific MNP signals, a detection limit down to a few nanogram MNP, in-vivo capability in conscious animals and measurement times of a few seconds, MRX is a valuable tool to improve the application of MNP for diagnostic and therapeutic purposes.

Identification of magnetic nanoparticles for combined positioning and lentiviral transduction of endothelial cells.

PURPOSE: The combination of magnetic nanoparticles (MNPs) with a magnetic field is a powerful approach to enable cell positioning and/or local gene therapy. Because physical requirements for MNPs differ between these two applications we have explored whether the use of different MNPs can provide site-specific positioning combined with efficient viral transduction of endothelial cells (ECs). METHODS: A variety of MNPs was screened for magnetic cell labeling and lentivirus binding. Then two different MNPs were chosen and their combined application was evaluated regarding EC magnetization and transduction efficiency. RESULTS: The combined use of PEI-Mag2 and NDT-Mag1 particles provided both efficient lentiviral transduction and high magnetic responsiveness of ECs that could be even retained within the vascular wall under flow conditions. The use of these MNPs did not affect biological characteristics of ECs like surface marker expression and vascular network formation. Importantly, with this method we could achieve an efficient functional overexpression of endothelial nitric oxide synthase in ECs. CONCLUSIONS: The application of two different MNPs provides optimal results for magnetic labeling of ECs in combination with viral transduction. This novel approach could be very useful for targeted gene therapy ex vivo and site-specific cell replacement in the vascular system.

Silica-iron oxide magnetic nanoparticles modified for gene delivery: a search for optimum and quantitative criteria.

PURPOSE: To optimize silica-iron oxide magnetic nanoparticles with surface phosphonate groups decorated with 25-kD branched polyethylenimine (PEI) for gene delivery. METHODS: Surface composition, charge, colloidal stabilities, associations with adenovirus, magneto-tranduction efficiencies, cell internalizations, in vitro toxicities and MRI relaxivities were tested for the particles decorated with varying amounts of PEI. RESULTS: Moderate PEI-decoration of MNPs results in charge reversal and destabilization. Analysis of space and time resolved concentration changes during centrifugation clearly revealed that at >5% PEI loading flocculation gradually decreases and sufficient stabilization is achieved at >10%. The association with adenovirus occurred efficiently at levels over 5% PEI, resulting in the complexes stable in 50% FCS at a PEI-to-iron w/w ratio of ≥7%; the maximum magneto-transduction efficiency was achieved at 9-12% PEI. Primary silica iron oxide nanoparticles and those with 11.5% PEI demonstrated excellent r(2)* relaxivity values (>600 s(-1)(mM Fe)(-1)) for the free and cell-internalized particles. CONCLUSIONS: Surface decoration of the silica-iron oxide nanoparticles with a PEI-to-iron w/w ratio of 10-12% yields stable aqueous suspensions, allows for efficient viral gene delivery and labeled cell detection by MRI.

Asymmetric flow field-flow fractionation of superferrimagnetic iron oxide multicore nanoparticles.

Magnetic nanoparticles are very useful for various medical applications where each application requires particles with specific magnetic properties. In this paper we describe the modification of the magnetic properties of magnetic multicore nanoparticles (MCNPs) by size dependent fractionation. This classification was carried out by means of asymmetric flow field-flow fractionation (AF4). A clear increase of the particle size with increasing elution time was confirmed by multi-angle laser light scattering coupled to the AF4 system, dynamic light scattering and Brownian diameters determined by magnetorelaxometry. In this way 16 fractions of particles with different hydrodynamic diameters, ranging between around 100 and 500 nm, were obtained. A high reproducibility of the method was confirmed by the comparison of the mean diameters of fractions of several fractionation runs under identical conditions. The hysteresis curves were measured by vibrating sample magnetometry. Starting from a coercivity of 1.41 kA m(-1) for the original MCNPs the coercivity of the particles in the different fractions varied from 0.41 to 3.83 kA m(-1). In our paper it is shown for the first time that fractions obtained from a broad size distributed MCNP fluid classified by AF4 show a strong correlation between hydrodynamic diameter and magnetic properties. Thus we state that AF4 is a suitable technology for reproducible size dependent classification of magnetic multicore nanoparticles suspended as ferrofluids.

Combined targeting of lentiviral vectors and positioning of transduced cells by magnetic nanoparticles.

Targeting of viral vectors is a major challenge for in vivo gene delivery, especially after intravascular application. In addition, targeting of the endothelium itself would be of importance for gene-based therapies of vascular disease. Here, we used magnetic nanoparticles (MNPs) to combine cell transduction and positioning in the vascular system under clinically relevant, nonpermissive conditions, including hydrodynamic forces and hypothermia. The use of MNPs enhanced transduction efficiency of endothelial cells and enabled direct endothelial targeting of lentiviral vectors (LVs) by magnetic force, even in perfused vessels. In addition, application of external magnetic fields to mice significantly changed LV/MNP biodistribution in vivo. LV/MNP-transduced cells exhibited superparamagnetic behavior as measured by magnetorelaxometry, and they were efficiently retained by magnetic fields. The magnetic interactions were strong enough to position MNP-containing endothelial cells at the intima of vessels under physiological flow conditions. Importantly, magnetic positioning of MNP-labeled cells was also achieved in vivo in an injury model of the mouse carotid artery. Intravascular gene targeting can be combined with positioning of the transduced cells via nanomagnetic particles, thereby combining gene- and cell-based therapies.

Cell Tracking by Magnetic Particle Imaging: Methodology for Labeling THP-1 Monocytes with Magnetic Nanoparticles for Cellular Imaging.

Magnetic particle imaging (MPI) is a noninvasive tomographic imaging modality for the quantitative visualization of magnetic nanoparticles (MNPs) with high temporal and spatial resolution. The general capability of MPI for cell tracking (e.g., monitoring living cells labeled with MNPs) has successfully been shown. MNPs in cell culture media are often subjected to structural and magnetic changes. In addition to the deteriorating reproducibility, this also complicates the systematic study of the relationship between the MNP properties and their cellular uptake for MPI. Here, we present a method for the preparation of magnetically labeled THP-1 (Tamm-Horsfall Protein-1) monocytes that are used in MPI cell tracking. The method development was performed using two different MPI tracers, which exhibited electrostatic and steric stabilizations, respectively. In the first step, the interaction between the MNPs and cell culture media was investigated and adjusted to ensure high structural and magnetic stability. Furthermore, the influences of the incubation time, MNP concentration used for cellular uptake, and individual preparation steps (e.g., the washing of cells) were systematically investigated. Finally, the success of the developed loading method was demonstrated by the MPI measurements. The presented systematic investigation of the factors that influence the MNP loading of cells will help to develop a reliable and reproducible method for MPI monocyte tracking for the early detection of inflammation in the future.

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.

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.

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.

Cellular effects of paclitaxel-loaded iron oxide nanoparticles on breast cancer using different 2D and 3D cell culture models.

BACKGROUND: Magnetic drug targeting (MDT) is an effective alternative for common drug applications, which reduces the systemic drug load and maximizes the effect of, eg, chemotherapeutics at the site of interest. After the conjugation of a magnetic carrier to a chemotherapeutic agent, the intra-arterial injection into a tumor-afferent artery in the presence of an external magnetic field ensures the accumulation of the drug within the tumor tissue. MATERIALS AND METHODS: In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) coated with lauric acid and human serum albumin as carriers for paclitaxel (SPIONLA-HSA-Ptx). To investigate whether this particle system is suitable for a potential treatment of cancer, we investigated its physicochemical properties by dynamic light scattering, ζ potential measurements, isoelectric point titration, infrared spectroscopy, drug release quantification, and magnetic susceptibility measurements. The cytotoxic effects were evaluated using extensive toxicological methods using flow cytometry, IncuCyte® live-cell imaging, and growth experiments on different human breast cancer cell lines in two- and three-dimensional cell cultures. CONCLUSION: The data showed that next to their high magnetization capability, SPIONLA-HSA-Ptx have similar cytostatic effects on human breast cancer cells as pure paclitaxel, suggesting their usage for future MDT-based cancer therapy.

Quantification of specific bindings of biomolecules by magnetorelaxometry.

The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP), to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX). Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated) serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality.The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

Combining Bulk Temperature and Nanoheating Enables Advanced Magnetic Fluid Hyperthermia Efficacy on Pancreatic Tumor Cells.

Many efforts are made worldwide to establish magnetic fluid hyperthermia (MFH) as a treatment for organ-confined tumors. However, translation to clinical application hardly succeeds as it still lacks of understanding the mechanisms determining MFH cytotoxic effects. Here, we investigate the intracellular MFH efficacy with respect to different parameters and assess the intracellular cytotoxic effects in detail. For this, MiaPaCa-2 human pancreatic tumor cells and L929 murine fibroblasts were loaded with iron-oxide magnetic nanoparticles (MNP) and exposed to MFH for either 30 min or 90 min. The resulting cytotoxic effects were assessed via clonogenic assay. Our results demonstrate that cell damage depends not only on the obvious parameters bulk temperature and duration of treatment, but most importantly on cell type and thermal energy deposited per cell during MFH treatment. Tumor cell death of 95% was achieved by depositing an intracellular total thermal energy with about 50% margin to damage of healthy cells. This is attributed to combined intracellular nanoheating and extracellular bulk heating. Tumor cell damage of up to 86% was observed for MFH treatment without perceptible bulk temperature rise. Effective heating decreased by up to 65% after MNP were internalized inside cells.

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.