Michael Hodenius

Magnetoliposomes: versatile innovative nanocolloids for use in biotechnology and biomedicine

The high biocompatibility and versatile nature of liposomes have made these particles keystone components in many hot-topic biomedical research areas. Liposomes can be combined with a large variety of nanomaterials, such as superparamagnetic iron oxide nanocores. Because the unique features of both the magnetizable colloid and the versatile lipid bilayer can be joined, the resulting so-called magnetoliposomes can be exploited in a great array of biotechnological and biomedical applications. In this article, we highlight the use of magnetoliposomes in immobilizing enzymes, both water-soluble and hydrophobic ones, as well as their potential in several biomedical applications, including MRI, hyperthermia cancer treatment and drug delivery. The goal of this article is not to list all known uses of magnetoliposomes but rather to present some conspicuous applications in comparison to other currently used nanoparticles.

FMN-coated fluorescent iron oxide nanoparticles for RCP-mediated targeting and labeling of metabolically active cancer and endothelial cells.

Riboflavin is an essential vitamin for cellular metabolism and is highly upregulated in metabolically active cells. Consequently, targeting the riboflavin carrier protein (RCP) may be a promising strategy for labeling cancer and activated endothelial cells. Therefore, Ultrasmall SuperParamagnetic Iron Oxide nanoparticles (USPIO) were adsorptively coated with the endogenous RCP ligand flavin mononucleotide (FMN), which renders them target-specific and fluorescent. The core diameter, surface morphology and surface coverage of the resulting FMN-coated USPIO (FLUSPIO) were evaluated using a variety of physico-chemical characterization techniques (TEM, DLS, MRI and fluorescence spectroscopy). The biocompatibility of FLUSPIO was confirmed using three different cell viability assays (Trypan blue staining, 7-AAD staining and TUNEL). In vitro evaluation of FLUSPIO using MRI and fluorescence microscopy demonstrated high labeling efficiency of cancer cells (PC-3, DU-145, LnCap) and activated endothelial cells (HUVEC). Competition experiments (using MRI and ICP-MS) with a 10- and 100-fold excess of free FMN confirmed RCP-specific uptake of the FLUSPIO by PC-3 cells and HUVEC. Hence, RCP-targeting via FMN may be an elegant way to render nanoparticles fluorescent and to increase the labeling efficacy of cancer and activated endothelial cells. This was shown for FLUSPIO, which due to their high T(2)-relaxivity, are favorably suited for MR cell tracking experiments and cancer detection in vivo.

A Concept for Magnetic Resonance Visualization of Surgical Textile Implants

Purpose:To develop a method for visualizing surgical textile implant (STI) with superparamagnetic iron oxides (SPIO), using magnetic resonance imaging (MRI). Therefore, positive-contrast inversion-recovery with on-resonant water suppression (IRON) was applied and its properties were evaluated in vitro. Materials and Methods:STI with different concentrations of SPIO integrated into the base material were produced. Imaging was performed on a clinical 1.5 Tesla scanner, using conventional balanced gradient echo sequences (SSFP), T2*-weighted sequences, and IRON-imaging. In vitro experiments were conducted in an agarose phantom. On MR-images, contrast-to-noise-ratios, and the dimensions of the implant were assessed. Results:Conventional MRI exhibited SPIO-loaded STI as signal voids. Using IRON, the mesh was clearly exhibited hyperintensely with suppression of on-resonant background signals with a distinct differentiation to other sources of off-resonances. Concentrations of approximately 9 mg/g led to best positive contrast and highest contrast-to-noise-ratios using IRON. Depending on B0-orientation, phase encoding direction and the STIs SPIO-load, the IRON-signal showed a characteristic pattern and an overestimation of STI size up to 4.6 mm. Conclusion:The integration of SPIOs into the base material combined with IRON is a feasible approach to visualize STI with MRI. This method could help to identify mesh-related problems in time and to reduce the need for surgical revision.

Synthesis of magnetic Fe3O4 particles covered with a modifiable phospholipid coat

This work reports the synthesis of iron oxide cores by coprecipitation of Fe2+ and Fe3+ ions with NaHCO3 or NH3. Depending on the experimental conditions, particles of two different sizes (13 or 130 nm diameter) were produced. X-ray diffractometry revealed Fe3O4 (magnetite) to be the main constituent. The smaller particles, which, in contrast to the larger ones, are superparamagnetic, were stabilized with a phospholipid bilayer consisting of a 9:1 molar ratio of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, thereby creating so-called magnetoliposomes. In a subsequent step, poly(ethylene glycol)-(PEG-) derivatized dipalmitoylphosphatidylethanolamine was introduced into the lipid envelope by incubating the magnetoliposomes with pre-formed sonicated vesicles containing the PEGylated lipid. The mechanism by which lipid transfer occurred was determined from the kinetic profiles. The relevance of these observations to a wide range of biomedical applications is briefly discussed.

Biotinylated Stealth® magnetoliposomes

Dimyristoylphosphatidylethanolamine (DC(14:0)PE) and the dioleoyl analogue (DC(18:1cis)PE) were mixed with alpha-biotinylamido-omega-N-succinimidoxycarbonyl-poly(ethylene glycol) (NHS-PEG-biotin) and quantitatively converted to alpha-biotinylamido-omega-(dimyristoylphosphatidylethanolamino-carbonyl)polyethylene glycol (DC(14:0)PE-PEG-biotin) and the dioleoyl analogue DC(18:1cis)PE-PEG-biotin, respectively. As shown by thin-layer chromatography and 1H NMR spectroscopy, PEGylation of both phosphatidylethanolamine types went to completion if the reaction was performed in organic solvent in the presence of triethylamine. The resulting derivatives were successfully incorporated into both classical phospholipid vesicles and a phospholipid bilayer surrounding nanometer-sized magnetite cores. In the latter case, the so-called activated Stealth(1) magnetoliposomes were produced which very efficiently immobilized streptavidinylated alkaline phosphatase.

Myocardial T(2) (*) mapping free of distortion using susceptibility-weighted fast spin-echo imaging: A feasibility study at 1.5 T and 3.0 T

This study demonstrates the feasibility of applying free‐breathing, cardiac‐gated, susceptibility‐weighted fast spin‐echo imaging together with black blood preparation and navigator‐gated respiratory motion compensation for anatomically accurate T  2* mapping of the heart. First, T  2* maps are presented for oil phantoms without and with respiratory motion emulation (T  2* = (22.1 ± 1.7) ms at 1.5 T and T  2* = (22.65 ± 0.89) ms at 3.0 T). T  2* relaxometry of a ferrofluid revealed relaxivities of R  2* = (477.9 ± 17) mM−1s−1 and R  2* = (449.6 ± 13) mM−1s−1 for UFLARE and multiecho gradient‐echo imaging at 1.5 T. For inferoseptal myocardial regions mean T  2* values of 29.9 ± 6.6 ms (1.5 T) and 22.3 ± 4.8 ms (3.0 T) were estimated. For posterior myocardial areas close to the vena cava T  2* ‐values of 24.0 ± 6.4 ms (1.5 T) and 15.4 ± 1.8 ms (3.0 T) were observed. The merits and limitations of the proposed approach are discussed and its implications for cardiac and vascular T  2* ‐mapping are considered. Magn Reson Med, 2009.

Polyelectrolyte coating of iron oxide nanoparticles for MRI-based cell tracking

UNLABELLED Iron oxide-based magnetic nanoparticles (MNPs) offer unique properties for cell tracking by magnetic resonance imaging (MRI) in cellular immunotherapy. In this study, we investigated the uptake of chemically engineered NPs into antigen-presenting dendritic cells (DCs). DCs are expected to perceive MNPs as foreign antigens, thus exhibiting the capability to immunologically sense MNP surface chemistry. To systematically evaluate cellular uptake and T2/T2(⁎) MR imaging properties of MNPs, we synthesized polymer-based MNPs by employing layer-by-layer (LbL) technology. Thereby, we achieved modification of particle shell parameters, such as size, surface charge, and chemistry. We found that subcellular packaging of MNPs rather than MNP content in DCs influences MR imaging quality. Increased local intracellular electron density as inferred from transmission electron microscopy (TEM) strongly correlated with enhanced contrast in MRI. Thus, LbL-tailoring of MNP shells using polyelectrolytes that impact on uptake and subcellular localization can be used for modulating MR imaging properties. FROM THE CLINICAL EDITOR In this study, layer-by-layer tailoring of magnetic NP shells was performed using polyelectrolytes to improve uptake by dendritic cells for cell-specific MR imaging. The authors conclude that polyelectrolyte modified NP-s can be used for modulating improving MR imaging quality by increasing subcellular localization.

In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes

The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

Fluorescent magnetoliposomes as a platform technology for functional and molecular MR and optical imaging

Here, we present a detailed characterisation of rhodamine B-containing magnetoliposomes (FLU-ML), emphasising the dependence of their fluorescence properties on the presence of iron oxide cores, and the molar fraction of the fluorophore. The magnetoliposome types used exist as colloidally stable, negatively charged clusters with an average hydrodynamic diameter of 95 nm. The molar rhodamine B fractions were 0.67 % and 1.97 %. Rhodamine B normalised fluorescence, quantum yields and fluorescence lifetimes were substantially reduced by inner filter effects as the magnetoliposome concentration is increased, by increasing molar rhodamine B fraction, and by quenching originating from the iron oxide cores. MR relaxometry at 3 T revealed extremely high r2 relaxivities (440 to 554 s-1mM-1) and moderately high r1 values (2.06 to 3.59 s-1mM-1). Upon incubating human prostate carcinoma (PC-3) cells with FLU-ML, a dose-dependent particle internalisation was found by MR relaxometry. In addition, the internalised FLU-ML were clearly visible by fluorescence microscopy. At the FLU-ML concentrations used (up to 3 × 10³ M Fe) cell viability was not substantially impaired. These results provide valuable insights on the fluorescence properties of bimodal magnetoliposomes and open promising perspectives for the use of these materials as a platform technology for advanced functional and molecular MR and optical imaging applications.

Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes

This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC(14:0)PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g(-1) Fe(3)O(4) and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r(2) relaxivities and moderately high r(1) values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.