Uptake of magnetic nanoparticles into cells for cell tracking

A challenge for future applications in nanotechnology is the functional integration of nano-sized materials into cellular structures. Here we investigated superparamagnetic Fe₃O₄ iron oxide nanoparticles coated with a lipid bilayer for uptake into cells and for targeting subcellular compartments. It was found that magnetic nanoparticles (MNPs) are effectively taken up into cells and make cells acquire magnetic activity. Biotin-conjugated MNPs were further functionalized by binding of the fluorescent tag streptavidin–fluorescein isothiocyanate (FITC) and, following uptake into cells, shown to confer magnetic activity and fluorescence labeling. Such FITC-MNPs were localized in the lysosomal compartment of cells which suggests a receptor-mediated uptake mechanism.     

Preparation of size-controlled nanoparticles of magnetite

Samples of ferrofluids containing chemically stabilized nanoparticles of magnetite (Fe₃O₄) with tetramethylammonium hydroxide (TMAOH) were prepared by a direct reduction–precipitation method. The influences of aging time and temperature on the size and monodispersion characteristics of the produced nanoparticles were investigated. Transmission electron microscopy, powder X-ray diffraction, Fourier-transform infrared, and magnetization measurements with applied magnetic field up to 2 T were used to characterize the synthesized iron oxides. Raising the temperature of the synthesized material in autoclave affects positively the monodispersion of the nanoparticles, but it was not found to significantly influence the size itself of individual particles.     

Enhancing remote controlled heating characteristics in hydrophilic magnetite nanoparticles via facile co-precipitation

Citric acid coated magnetite nanoparticles were synthesized using a one-step and two-step co-precipitation method at different temperatures. The stability of the nanoparticles in aqueous media was compared. The magnetic heating characteristics in an alternating magnetic field were examined and specific absorption rates were determined. The nanoparticles were characterized by various techniques (Fourier transform infrared, UV spectrophotometry, thermogravimetric analysis, dynamic light scattering, transmission electron microscopy, X-ray diffraction and vibrating sample magnetometry). The temperature of synthesis and mode of functionalizing the particles affected their physical and magnetic properties. Higher temperatures led to increased specific absorption rates for both methods but more stable hydrophilic superparamagnetic nanoparticles were obtained in the one-step method.     

MR tracking of magnetically labeled mesenchymal stem cells in rats with liver fibrosis

Purpose

In vivo magnetic resonance (MR) tracking of magnetically labeled bone marrow mesenchymal stem cells (BMSCs) administered via the mesenteric vein to rats with liver fibrosis.

Materials and Methods

Rat BMSCs were labeled with superparamagnetic iron oxide (SPIO) and the characteristics of the BMSCs after labeling were investigated. Eighteen rats with CCL4-induced liver fibrosis were randomized to three groups to receive SPIO-labeled BMSCs (BMSC-labeled group), cell-free SPIO (SPIO group), or unlabeled BMSCs (control group). MR imaging of the liver was performed at different time points, and signal-to-noise ratio (SNR) of the liver was measured. In vivo distribution of delivered BMSCs was assessed by histological analysis.

Results

Labeling of BMSCs with SPIO did not significantly alter cell viability and proliferation activity. In BMSC-labeled group, the liver SNR immediately decreased from 8.56±0.26 to 3.53±0.41 at 1 h post injection and remained at a significantly lower level till 12 days (P<.05 versus the level before). By contrast, the liver SNR of the SPIO group almost recovered to the preinjection level (P=.125) at 3 days after a transient decrease. In control group, the liver SNR demonstrated no significant difference at the tested time points. Additionally, Prussian blue-positive cells were mainly distributed in the liver parenchyma, especially in injured areas.

Conclusion

The magnetically labeled BMSCs infused through the mesenteric vein can be detected in the fibrotic liver of rats using in vivo MR imaging up to 12 days after injection.     

Formation and properties of magnetic chains for 100 nm nanoparticles used in separations of molecules and cells

Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations 10¹⁰/mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immuno-magnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.     

Labeling of macrophages using bacterial magnetosomes and their characterization by magnetic resonance imaging

This work investigated macrophages labeled with magnetosomes for the possible detection of inflammations by MR molecular imaging. Pure magnetosomes and macrophages containing magnetosomes were analyzed using a clinical 1.5 T MR-scanner. Relaxivities of magnetosomes and relaxation rates of cells containing magnetosomes were determined. Peritonitis was induced in two mice. T₁, T₂ and T₂* weighted images were acquired following injection of the probes. Pure magnetosomes and labeled cells showed slight effects on T₁, but strong effects on T₂ and T₂* images. Labeled macrophages were located with magnetic resonance imaging (MRI) in the colon area, thus demonstrating the feasibility of the proposed approach.     

Optimization of nanoparticle core size for magnetic particle imaging

Magnetic particle imaging (MPI) is a powerful new research and diagnostic imaging platform that is designed to image the amount and location of superparamagnetic nanoparticles in biological tissue. Here, we present mathematical modeling results that show how MPI sensitivity and spatial resolution both depend on the size of the nanoparticle core and its other physical properties, and how imaging performance can be effectively optimized through rational core design. Modeling is performed using the properties of magnetite cores, since these are readily produced with a controllable size that facilitates quantitative imaging. Results show that very low detection thresholds (of a few nanograms Fe₃O₄) and sub-millimeter spatial resolution are possible with MPI.     

Examination of magnetite nanoparticles utilising the temperature dependent magnetorelaxometry

We investigated the magnetic properties of two kinds of magnetosomes (25-42 nm) produced by the magneto-tactic bacterium Magnetospirillum gryphiswaldense. At temperatures between 4.2 K and room temperature the temperature dependent magnetorelaxation (TMRX) method was used. We found three areas with magnetic signals and discuss them in this paper using the results of additional hysteresis loop measurements. The signals detected above 300 K show the lower end of a beginning energy barrier distribution. The signals between 70 and 120 K lie in the area of the Verwey transition and disappear over time due to aging processes. In addition to these signals, other signals at temperatures between 4.2 and 70 K were found and possible causes are discussed.     

Electro-precipitation of Fe3O4 nanoparticles in ethanol

The preparation of superparamagnetic magnetite (Fe₃O₄) nanoparticles by electro-precipitation in ethanol is proposed. Particle average size can be set from 4.4 to 9 nm with a standard deviation around 20%. Combination of wide-angle X-ray scattering (WAXS), Electron energy loss spectroscopy (EELS) and Mössbauer spectroscopy characterizations clearly identifies the particles as magnetite single-crystals (Fe₃O₄).     

Synthesis of magnetic rhenium sulfide composite nanoparticles

Rhenium sulfide nanoparticles are associated with magnetic iron oxide through coprecipitation of iron salts with tetramethylammonium hydroxide. Sizes of the formed magnetic rhenium sulfide composite particles are in the range 5.5-12.5 nm. X-ray diffraction and energy-dispersive analysis of X-rays spectra demonstrate the coexistence of Fe₃O₄ and ReS₂ in the composite particle, which confirm the formation of the magnetic rhenium sulfide composite nanoparticles. The association of rhenium sulfide with iron oxide not only keeps electronic state and composition of the rhenium sulfide nanoparticles, but also introduces magnetism with the level of 24.1 emu g^-1 at 14 kOe. Surface modification with monocarboxyl-terminated poly(ethylene glycol) (MPEG-COOH) has the role of deaggregating the composite nanoparticles to be with average hydrodynamic size of 27.3 nm and improving the dispersion and the stability of the composite nanoparticles in water.     

Instant magnetic labeling of tumor cells by ultrasound in vitro

Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.     

Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled

Objective

Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated.

Methods

After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied.

Results

Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2 ± 118.8, 2031.9 ± 59.7 and 1887.4 ± 40.8 measured by MRI. Differences between these three groups were statistically significant (P < 0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178 ± 0.009 mg/L, 0.158 ± 0.003 mg/L and 0.120 ± 0.002 mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P < 0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5 × 10⁴ Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12 days.

Conclusions

Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPA-labeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible.     

Cellular interactions of lauric acid and dextran-coated magnetite nanoparticles

In vitro cytocompatibility and cellular interactions of lauric acid and dextran-coated magnetite nanoparticles were evaluated with two different cell lines (mouse fibroblast and human cervical carcinoma). Lauric acid-coated magnetite nanoparticles were less cytocompatible than dextran-coated magnetite nanoparticles and cellular uptake of lauric acid-coated magnetic nanoparticles was more than that of dextran-coated magnetite nanoparticles. Lesser cytocompatibility and higher uptake of lauric acid-coated magnetite nanoparticles as compared to dextran-coated magnetic nanoparticles may be due to different cellular interactions by coating material. Thus, coating plays an important role in modulation of biocompatibility and cellular interaction of magnetic nanoparticles.     

Simple and facile approach to synthesize magnetite nanoparticles and assessment of their effects on blood cells

In this paper, a very simple and facile approach for the large scale synthesis of uniform and size-controllable single-domain magnetite nanoparticles is reported. These magnetite nanoparticles were synthesized via thermal decomposition of a ferric nitrate/ethylene glycol solution. The structural and morphological properties of the synthesized nanoparticles were carefully studied. Nearly spherical nanoparticles with inverted spinel structure and average particle and crystallite sizes smaller than 20 nm were obtained. The magnetic measurements revealed that magnetite nanoparticles have a magnetic saturation value near that of the bulk magnetite. The erythrocyte cytotoxicity assays showed no hemolytic potential of the samples containing magnetite nanoparticles, indicating no cytotoxic activity on human erythrocytes, which makes these interesting for biotechnological applications.     

Tracking of transplanted mesenchymal stem cells labeled with fluorescent magnetic nanoparticle in liver cirrhosis rat model with 3-T MRI

In vivo visualization of transplanted stem cells with noninvasive technique is essential for the monitoring of cell implantation, homing and differentiation. At present, superparamagnetic iron oxide (SPIO) is most commonly used for cell labeling. However, stem cells lack phagocytic capacity and transfection agent is required for sufficient internalization of SPIO for cellular imaging. However, the potential hazards of transfection agents are not fully investigated. Instead of SPIO, we used commercially available new tagging material, fluorescent magnetic nanoparticle (MNP) containing rhodamine B isothiocyanate within a silica shell (Biterials, Seoul, Korea). This tagging material does not require transfection agents for the cell labeling. In addition to that, the core of this MNP is composed of ferrite and the inner portion of silica shell contains fluorescent materials, therefore, it has both magnetic and optical features. This study was designed to track intrasplenically injected bone marrow mesenchymal stem cells (MSCs) labeled with fluorescent MNP in liver cirrhosis rat model with 3-T magnetic resonance equipment. We compared magnetic resonance imaging (MRI) of livers in rats which were injected with non-labeled stem cells or labeled stem cells with MNP or SPIO. We found that the respective liver-to-muscle contrast-to-noise ratios at 3 and 5 h after MNP or SPIO-labeled stem cell injection was significantly lower than that of pre-injection and non-labeled group. There was no significant difference between MNP-labeled group and SPIO-labeled group. We can effectively detect intrasplenically injected MNP-labeled MSCs in an experimental rat model of liver cirrhosis with 3-T MRI.     

Cytotoxicity and GMI bio-sensor detection of maghemite nanoparticles internalized into cells

In this work we determine conditions to produce cell samples for imaging with detection of the modification of the magnetic field by maghemite (Fe₂O₃) nanoparticles acting as a high sensitivity magnetic bio-sensor based on the giant magneto-impedance (GMI) effect. Mat Ly Lu cells are grown for 24 h with various maghemite nanoparticles concentrations (from 0 to 6 mg/ml). The percentage of viable cells is determined by counting labeled cells with trypan blue under an optical microscope. The quantity of nanoparticles internalized into the cells is evaluated by X-ray fluorescence analysis and expressed in iron moles per cell. The GMI bio-sensor was tested with the various samples. We observed that the best sensitivity of the GMI bio-sensor was obtained at a frequency of 1 MHz. To confirm these results in the presence of cell samples, four measurement frequencies were pre-selected (from 1 to 100 MHz) and tested. Cell growth conditions compatible with an acceptable percentage of cell viability for various concentrations of nanoparticles were also determined. These experiments allow us to conclude that cell growth with 0.1 mg/ml of nanoparticles for 24 h shows modifications of the magnetic field detectable optimally at 1 MHz frequency.     

Magneto-optical study of magnetite nanoparticles prepared by chemical and biomineralization process

This paper deals with a magneto-optical study of suspensions of magnetosomes. These magnetosomes are synthesized by biomineralization process of magnetotactic bacteria, followed by steps of isolation and purification in order to obtain stable suspensions. The structural analysis evidences the good crystallinity of the magnetite particles with a diameter of 34 nm. Magneto-induced linear and circular anisotropy confirms the important role played by the chains in the orientation mechanism of such magnetic dipoles. Numerical adjustments of the linear anisotropy curves using a classical Langevin orientation model give the average number of magnetosomes per chain, about 12.     

Application of citrate-stabilized gold-coated ferric oxide composite nanoparticles for biological separations

Gold-coated magnetic nanoparticles were synthesized with size ranging from 15 to 40 nm using sodium citrates as the reducing agent. Oxidized magnetites (Fe₃O₄) fabricated by co-precipitation of Fe²⁺ and Fe³⁺ in strong alkaline solution were used as magnetic cores. The structures of gold (Au) shell and magnetic core (Au–Fe) were studied by transmission electron microscopy (TEM) image and energy dispersive spectroscopy (EDS) spectrum. Results from high-resolution X-ray diffraction (HR XRD) show that the Au–Fe oxide nanoparticles have a face-centered cubic shape with the crystalline faces of {1 1 1}. The Au-coated magnetic nanoparticles exhibited a surface plasmon resonance peak at 528 nm. The nanoparticles are well dispersed in distilled water. A 3000 G permanent magnet was successfully used for the separation of the functionalized nanoparticles. Magnetic properties of the nanoparticles were determined by magnetic force microscope (MFM) in nanometric resolution and vibrating sample magnetometer (VSM). Magnetic separation of biological molecules using Au-coated magnetic oxide composite nanoparticles was examined after attachment of protein immunoglobulin G (IgG) through electrostatic interactions. Using this method, separation was achieved with a maximum yield of 35% at an IgG concentration of 400 ng/ml.