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.

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.

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

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.

Selection of potential iron oxide nanoparticles for breast cancer treatment based on in vitro cytotoxicity and cellular uptake.

Superparamagnetic iron oxide nanoparticles (SPIONs) are promising tools for the treatment of different diseases. Their magnetic properties enable therapies involving magnetic drug targeting (MDT), hyperthermia or imaging. Depending on the intended treatment, specific characteristics of SPIONs are required. While particles used for imaging should circulate for extended periods of time in the vascular system, SPIONs intended for MDT or hyperthermia should be accumulated in the target area to come into close proximity of, or to be incorporated into, specific tumor cells. In this study, we determined the impact of several accurately characterized SPION types varying in size, zeta potential and surface coating on various human breast cancer cell lines and endothelial cells to identify the most suitable particle for future breast cancer therapy. We analyzed cellular SPION uptake, magnetic properties, cell proliferation and toxicity using atomic emission spectroscopy, magnetic susceptometry, flow cytometry and microscopy. The results demonstrated that treatment with dextran-coated SPIONs (SPIONDex) and lauric acid-coated SPIONs (SPIONLA) with an additional protein corona formed by human serum albumin (SPIONLA-HSA) resulted in very moderate particle uptake and low cytotoxicity, whereas SPIONLA had in part much stronger effects on cellular uptake and cellular toxicity. In summary, our data show significant dose-dependent and particle type-related response differences between various breast cancer and endothelial cells, indicating the utility of these particle types for distinct medical applications.

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.

Magnetic nanoparticles adapted for specific biomedical applications.

Magnetic nanoparticles (MNPs) are used in different biomedical applications, whereby each application requires specific particle properties. To fulfill these requirements, particle properties have to be optimized by means of variation of crystal structure, particle size, and size distribution. To this aim, improved aqueous precipitation procedures for magnetic iron oxide nanoparticle synthesis were developed. One procedure focused on the cyclic growth of MNPs without nucleation of new particle cores during precipitation. The second novel particle type are magnetic multicore nanoparticles, which consist of single cores of approximately 10 nm forming dense clusters in the size range from 40 to 80 nm. Their highest potential features these multicore particles in hyperthermia application. In our in vivo experiments, therapeutically suitable temperatures were reached after 20 s of heating for a particle concentration in the tumor of 1% and field parameters of H=24 kA/m and f=410 kHz. This review on our recent investigations for particle optimization demonstrates that tuning magnetic properties of MNPs can be obtained by the alteration of their structure, size, and size distribution. This can be realized by means of control of particle size during synthesis or subsequent size-dependent fractionation. The here-developed particles show high potential for biomedical applications.

Development of a lauric acid/albumin hybrid iron oxide nanoparticle system with improved biocompatibility.

The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 µg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical application. The synthesis is straightforward and reproducible and thus easily translatable into a good manufacturing practice environment.

Development and characterization of magnetic iron oxide nanoparticles with a cisplatin-bearing polymer coating for targeted drug delivery.

A highly selective and efficient cancer therapy can be achieved using magnetically directed superparamagnetic iron oxide nanoparticles (SPIONs) bearing a sufficient amount of the therapeutic agent. In this project, SPIONs with a dextran and cisplatin-bearing hyaluronic acid coating were successfully synthesized as a novel cisplatin drug delivery system. Transmission electron microscopy images as well as X-ray diffraction analysis showed that the individual magnetite particles were around 4.5 nm in size and monocrystalline. The small crystallite sizes led to the superparamagnetic behavior of the particles, which was exemplified in their magnetization curves, acquired using superconducting quantum interference device measurements. Hyaluronic acid was bound to the initially dextran-coated SPIONs by esterification. The resulting amide bond linkage was verified using Fourier transform infrared spectroscopy. The additional polymer layer increased the vehicle size from 22 nm to 56 nm, with a hyaluronic acid to dextran to magnetite weight ratio of 51:29:20. A maximum payload of 330 µg cisplatin/mL nanoparticle suspension was achieved, thus the particle size was further increased to around 77 nm with a zeta potential of -45 mV. No signs of particle precipitation were observed over a period of at least 8 weeks. Analysis of drug-release kinetics using the dialysis tube method revealed that these were driven by inverse ligand substitution and diffusion through the polymer shell as well as enzymatic degradation of hyaluronic acid. The biological activity of the particles was investigated in a nonadherent Jurkat cell line using flow cytometry. Further, cell viability and proliferation was examined in an adherent PC-3 cell line using xCELLigence analysis. Both tests demonstrated that particles without cisplatin were biocompatible with these cells, whereas particles with the drug induced apoptosis in a dose-dependent manner, with secondary necrosis after prolonged incubation. In conclusion, combination of dextran-coated SPIONs with hyaluronic acid and cisplatin represents a promising approach for magnetic drug targeting in the treatment of cancer.

Highly stable monodisperse PEGylated iron oxide nanoparticle aqueous suspensions: a nontoxic tracer for homogeneous magnetic bioassays.

Uniformly sized and shaped iron oxide nanoparticles with a mean size of 25 nm were synthesized via decomposition of iron-oleate. High resolution transmission electron microscopy and Mössbauer spectroscopy investigations revealed that the particles are spheres primarily composed of Fe3O4 with a small fraction of FeO. From Mössbauer and static magnetization measurements, it was deduced that the particles are superparamagnetic at room temperature. The hydrophobic particles were successfully transferred into water via PEGylation using nitrodopamine as an anchoring group. IR spectroscopy and thermogravimetric analysis showed the success and efficiency of the phase transfer reaction. After PEGylation, the particles retained monodispersity and their magnetic core remained intact as proven by photon cross-correlation spectroscopy, ac susceptibility, and transmission electron microscopy. The particle aqueous suspensions revealed excellent water stability over a month of monitoring and also against temperature up to 40 °C. The particles exhibited a moderate cytotoxic effect on in vitro cultured bone marrow-derived macrophages and no release of inflammatory or anti-inflammatory cytokines. The PEGylated particles were functionalized with Herceptin antibodies via a conjugation chemistry, their response to a rotating magnetic field was studied using a fluxgate-based setup and was compared with the one recorded for hydrophobic and PEGylated particles. The particle phase lag rose after labeling with Herceptin, indicating the successful conjugation of Herceptin antibodies to the particles.

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.

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.

Targeted delivery of magnetic aerosol droplets to the lung.

The inhalation of medical aerosols is widely used for the treatment of lung disorders such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory infection and, more recently, lung cancer. Targeted aerosol delivery to the affected lung tissue may improve therapeutic efficiency and minimize unwanted side effects. Despite enormous progress in optimizing aerosol delivery to the lung, targeted aerosol delivery to specific lung regions other than the airways or the lung periphery has not been adequately achieved to date. Here, we show theoretically by computer-aided simulation, and for the first time experimentally in mice, that targeted aerosol delivery to the lung can be achieved with aerosol droplets comprising superparamagnetic iron oxide nanoparticles--so-called nanomagnetosols--in combination with a target-directed magnetic gradient field. We suggest that nanomagnetosols may be useful for treating localized lung disease, by targeting foci of bacterial infection or tumour nodules.