Advanced Review

Magnetoliposomes as magnetic resonance imaging contrast agents Stefaan J. Soenen,1 Greetje Vande Velde,2 Ashwini Ketkar-Atre,2 Uwe Himmelreich2 and Marcel De Cuyper1∗ Among the wide variety in iron oxide nanoparticles which are routinely used as magnetic resonance imaging (MRI) contrast agents, magnetoliposomes (MLs) take up a special place. In the present work, the two main types (large and small MLs) are defined and their specific features are commented. For both types of MLs, the flexibility of the lipid coating allows for efficient functionalization, enabling bimodal imaging (e.g., MRI and fluorescence) or the use of MLs as theranostics. These features are especially true for large MLs, where several magnetite cores are encapsulated within a single large liposome, which were found to be highly efficient theranostic agents. By carefully fine-tuning the number of magnetite cores and attaching Gd3+ -complexes onto the liposomal surface, the large MLs can be efficiently optimized for dynamic MRI. A special type of MLs, biogenic MLs, can also be efficiently used in this regard, with potential applications in cancer treatment and imaging. Small MLs, where the lipid bilayer is immediately attached onto a solid magnetite core, give a very high r2 /r1 ratio. The flexibility of the lipid bilayer allows the incorporation of poly(ethylene glycol)–lipid conjugates to increase blood circulation times and be used as bone marrow contrast agents. Cationic lipids can also be incorporated, leading to high cell uptake and associated strong contrast generation in MRI of implanted cells. Unique for these small MLs is the high resistance the particles exhibit against intracellular degradation compared with dextran- or citrate-coated particles. Additionally, intracellular clustering of the iron oxide cores enhances negative contrast generation and enables longer tracking of labeled cells in time.  2011 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol 2011 3 197–211 DOI: 10.1002/wnan.122

INTRODUCTION

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ue to the relative insensitivity of magnetic resonance imaging (MRI, see Box 1), the use of contrast agents such as paramagnetic Gd3+ (referred to as a positive, T1 -contrast agent) or superparamagnetic iron oxide nanocores (a so-called negative, T2 -contrast agent) is a common practice to enhance detection limits and visualization of targeted organs or cells.1–4 Due to the high toxicity associated with Gd3+ , the use of chelates and an efficient renal clearance of the contrast agents is highly desirable.5,6 For in vitro cell labeling experiments or long-term ∗ Correspondence

to: [email protected]

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Lab of BioNanoColloids, KULeuven Campus Kortrijk, IRC Etienne Sabbelaan, Kortrijk, Belgium

2 Biomedical

NMR Unit/MoSAIC, KULeuven Campus Gasthuisberg, University Medical Hospital Gasthuisberg, Leuven, Belgium DOI: 10.1002/wnan.122

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in vivo cell tracking studies, the clearance of the particles should be far lower, which impedes the use of Gd3+ -based agents for these purposes. For these types of studies, superparamagnetic iron oxide nanoparticles (SPIOs) are most frequently used.7,8 As these iron oxide nanocores will be subjected to a biological environment, the surface of the metal oxide cores must be carefully tailored to prevent substantial aggregation of the nanocores, which might render them less suitable for most biomedical applications.9 In the literature, a wide variety of possible coating materials have been described, including—but not limited to—dextran (Resovist, Endorem/Feridex in the US, which have long been the standard MR contrast agents for liver lesions in clinical settings, but have recently been taken off the market10 ), citrate very small iron oxide particles (VSOP), polystyrene/divinylbenzenze (Bangs particles), silica, gold, dimercapto-succinic

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acid, functionalized polyethylene glycol (PEG), or lipids (magnetoliposomes, MLs).9,11–16

BOX 1 MAGNETIC RESONANCE IMAGING MRI has emerged as a powerful, noninvasive method in the field of molecular and cellular imaging. The high resolution and excellent soft tissue contrast are the main advantages over other in vivo imaging techniques. MR images of biological samples are most commonly generated from hydrogen atoms (more precisely from protons) of mobile molecules like water and lipids. Various methods exist to generate contrast in MRI to visualize tissue properties like concentration differences of water or lipids, tissue viscosity, flow, diffusion, iron content, chemical composition, and others. The longitudinal (T1 ) and transverse relaxation (T2 and T2 *) of protons are most commonly used mechanisms for contrast generation.17,18 For molecular and cellular MR imaging, contrast agents are used that modulate those relaxation times. Lanthanide chelates and manganesebased compounds generate hyperintense contrast in T1 -weighted MRI. They are often referred to as T1 -contrast agents; however, they also modulate T2 relaxation, which is often detected with higher sensitivity. Superparamagnetic compounds like iron-containing nanoparticles (NPs) cause highly sensitive changes in T2 /T2 * relaxation times and therefore hypointense signals in T2 /T2 *-weighted MRI methods. Protocols for the acquisition of MRI data for molecular and cellular imaging are similar to other routine MRI protocols.18 Protocols like T2 -weighted gradientecho sequences that emphasize signal loss due to field inhomogeneities are beneficial for ironoxide-based contrast agents. More specialized MRI techniques were recently developed to generate hyperintense (bright) contrast for the detection of iron-labeled cells.17 Magnetization transfer techniques are used to exploit contrast generated by CEST contrast agents.18 These compounds generate two or more pools of protons with a shift of their resonance frequencies. Hereby, different populations can be visualized simultaneously, leading potentially to ‘multi-color’ MRI. To overcome specificity problems of T2 /T2 *-based cell visualization, fluorinated contrast agents in combination with 19 F MRI has been used.19,20 The recent developments of hyperpolarization methods have

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resulted in an increase of sensitivity for otherwise relatively insensitive nuclei like 13 C and 15 N by 10,000-fold and more opening new perspectives for MRI-based molecular imaging.21

The general concept of MLs is largely based on the structure and specific features of well-known phospholipid vesicles. Liposomes are the most frequently employed carrier vehicles for drug delivery purposes, thanks to their dominant role due to the many outstanding properties they possess: (1) a high biocompatibility, (2) the possibility to undergo a wide variety in surface manipulations by chemical means, (3) the ability to enclose both hydrophilic (in the inner aqueous cavity) as well as hydrophobic (in the lipid bilayer) compounds, (4) the ability to simultaneously contain both imaging and therapeutic agents, and (5) additionally, the size of liposomes can be controlled to a relatively high degree, ranging from approximately 20 nm to over 1 µm.22 Liposomes with incorporated (ultrasmall, U) SPIOs are generally referred to as MLs.16 Thanks to the lipid coating, MLs can easily be functionalized by addition of PEG to enhance blood circulation times, enable active targeting to specific locations by peptide or antibody conjugation, or combine the magnetic properties of MLs with other imaging modalities such as optical imaging or tomography or include anticancer drugs.23,24 In general, two main types of MLs can be discerned as seen in Figure 1: (1) Large unilamellar vesicles—typically prepared by extrusion or organic solvent preparation—which encompass multiple water-soluble (U)SPIOs in the inner aqueous cavity (b) (a)

FIGURE 1 | (a) Transmission electron micrograph of small (cationic) magnetoliposomes (MLs) showing the phospholipid bilayer (visualized by negative staining using 2% uranylacetate) surrounding the electron dense iron oxide cores. (b) Cryo-electron micrograph of a large (cationic) ML showing dispersed nanosized, citrate-coated iron oxide cores within a unilamellar liposome. Scale bars: 50 nm. (Reprinted with permission from Ref 27. Copyright 2009 Elsevier.)

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of the liposome (called here ‘large MLs’). These particles possess the ability to encapsulate water-soluble molecules such as anticancer drugs and allow to precisely fine-tune the amount of iron oxide per liposome. (2) Small (14-nm-sized diameter) iron oxide cores where a lipid double layer is immediately attached to the surface of the iron oxide cores (here referred to as ‘small MLs’).25 Next to all the advantages of surface manipulations which are also possible for large MLs, small MLs also present several unique features which are mostly because of the unique formulation of the lipid bilayer surrounding the iron oxide nanocore. Here, the inner leaflet of the lipid bilayer is strongly chemisorbed to the iron oxide surface, whereas the outer leaflet of the lipid bilayer is more loosely physisorbed and can easily be functionalized.26 The excellent soft tissue contrast and high spatial resolution of MRI make it one of the most widely applied imaging methods in clinics and biomedical research. The use of imaging probes, such as SPIOs or Gd3+ -containing complexes, is generally required to further enhance the image contrast. To date, the number of different imaging probes is enormous, leading to a vast array of tools, each with their specific features. For iron oxide NPs, MLs have emerged as highly versatile and fascinating research tools which combine an efficient T2 -contrast generation with the advantages of a flexible liposome coating. The present work aims to provide an overview of the current state of art of MLs as MRI contrast agents. In the first part of the present work, an overview is given on the use of large MLs, with special attention being paid to multimodal imaging and the combination with therapeutic agents (Box 2). Then, a small part is included on bacterial-derived MLs as a special type of MR contrast agent with high potential in cancer treatment, imaging, and theranostics. In the final part, the unique properties of small MLs and the importance thereof as an MR contrast agent will be discussed in more detail.

LARGE MLS BOX 2 LIPID NOMENCLATURE The wide variety in types of phospholipids which can be used to generate MLs can be rather confusing and hinder a direct comparison of results between various studies employing different lipid compositions. In general, phospholipids can be classified under three main types depending on their overall charge at physiological conditions: zwitterionic, anionic, and cationic

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lipids. Zwitterionic and anionic lipids are typically amphiphilic phospholipids, containing two hydrophobic fatty acyl chains and a hydrophilic glycerol moiety with a phosphate group linked to a headgroup, which will determine the actual charge. Fatty acyl chains comprising 14 (myristoyl-), 16 (palmitoyl-), or 18 (oleoyl- [C18:1] or stearoyl- [C18:0]; mono-unsaturated vs fully saturated) carbons are often encountered. A typical anionic lipid is—phosphatidylglycerol (PG) which has a glycerol base in the polar headgroup. For zwitterionic lipids, the headgroup is usually choline (–PC; chemically inert quaternary ammonium group) or ethanolamine [(–PE); which contains a free amine group and is therefore easily modified by chemical means to include PEG, peptides, chelates, or fluorescent groups]. Cationic lipids are synthetic lipids and do not contain a phosphate moiety. These lipids are frequently used to enhance the efficiency of cell transfections with DNA.28 The most commonly used cationic lipids are: didecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DODAB), dioleoyltrimethylammonium propane (DOTAP), and distearoyltrimethylammonium propane (DSTAP).

EFFECTS OF LIPOSOMAL COATINGS ON CELL UPTAKE The use of liposomes to encapsulate iron oxide NPs and the advantages of such systems were already described in the early nineties. Bulte et al.29 found that when dextran-coated magnetite NPs were encapsulated within large unilamellar vesicles, these MLs could be used for the efficient labeling of human blood mononuclear cells, such as peripheral blood lymphocytes and monocytes. This resulted in an increase in R2 which was approximately threefold higher than what could be achieved using dextrancoated magnetite NPs functionalized with monoclonal antibodies. Furthermore, functionalizing the liposome surface by Sendai Virus particles did not lead to a further significantly enhanced cellular uptake, implicating that the lipid bilayer composition of the large MLs, as such, intrinsically possesses a high uptake tendency. One of the first advantages of liposomal coatings to be exploited was the size manipulation of the liposomes. Based on the different synthesis protocols, including thin film hydration, extrusion, or sonication, various types of liposomes ranging in size from a few

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tens of nanometers to nearly one micrometer can be prepared. The mean particle size is well controllable and depends on the applied conditions during synthesis and the type of lipids used. Because of this, the particle size is quite reproducible from batch to batch and the size of the liposome can be adjusted to better suit the desired application. This offers great advantages to other coating agents such as small molecules or polymers, which are generally quite restricted in terms of spatial manipulation and size control and lead only to one size of particles within a certain size distribution. In a study by Chan et al.30 four different types of coating for iron oxide particles (bovine serum albumin, the Fc fragment of IgG, dextran, and lipids) were compared with respect to the visualization of abscesses by MRI. It was found that lipid-coated iron oxide cores were superior to image abscesses by virtue of their phagocytosis in surrounding inflammatory cells. Furthermore, larger particles such as the lipid-encapsulated ones (90 nm diameter) were superior compared to smaller particles such as bovine serum albumin-coated ones (35 nm diameter) with respect to uptake in human buffy coat (61 ± 6.2% vs 6 ± 1.8%). In Sprague–Dawley rats with subcutaneous abscesses, only those animals injected intravenously with lipid-encapsulated particles resulted in a hypointense ring around the abscess, which could be confirmed by histological assessment, showing iron granules in phagocytic cells at the periphery of the abscesses. Taken together, the above studies pointed to a high, nonspecific uptake of MLs by virtue of their liposomal coating. Similar to other types of SPIOs, nonspecific interactions of the particles with the cell surface induce cellular internalization of the particles with a certain efficiency, depending on the extent of association of the particles with the cell surface and the endocytic capacity of the cell type used. The flexibility of the liposomal coating of MLs can facilitate this process, by inclusion of cationic lipids such as DOTAP which leads to significant increases in cell loading efficiency as noticed for human cervical cancer cells (HeLa), human prostatic adenocarcinoma cells (PC3), murine neuroblastoma cells (Neuro-2a), and murine colorectal adenocarcinoma cells (CT-26).31 In case specific uptake (by means of a cell-specific surface receptor) is envisioned, such a high nonspecific uptake would lead to a high background contrast, hampering any targeting efforts. Meincke et al.32 observed that the nonspecific uptake could be significantly impaired by the incorporation of PEGylated lipids. When using 5% 1,2-distearoyl-sn-glycero-3phosphoethanolamine (DSPE)-PEG2000, uptake of large MLs by human U343 glioma cells could be 200

reduced by 86%. A further increase of the amount of PEG (10 or 15%) did not have any additional effects. In an in vivo tumor model, it was shown that next to the presence of PEG, the number of MLs injected also contributed to remarkable differences in terms of reducing the relative signal intensity in adenocarcinoma in the liver of rats.33

LARGE MLS AS BIMODAL IMAGING AGENTS Large MLs were found to be efficient MR contrast agents, with the T2 -contrast enhancement being dependent on the liposomal iron load.34 For highly purified MLs, the ratio of transversal over longitudinal relaxivities of water protons in ML dispersions was between 6 and 18, which ranks them among the best T2 -contrast agents described to date.34 Furthermore, due to the magnetic properties of the MLs, magnetic targeting by means of an extracorporeal magnet above one flank of Swiss nude mice bearing a PC3 human prostate carcinoma tumor in each flank was found to be feasible, leading to a 52% contrast enhancement in the magnetically targeted tumor and 7% enhancement in the nontargeted tumor.35 As previously stated, the wide versatility in different lipid types and lipid conjugates has stimulated the development of bifunctional MLs. With regard to imaging, fluorescent lipid conjugates are most commonly incorporated in the lipid bilayer, allowing both MRI and fluorescence detection of the MLs. When these liposomes are further functionalized with recombinant annexin A5 molecules, this enabled the detection of the apoptotic T-lymphoma cell line (Jurkat cells) by both optical techniques and MRI.36 This bimodal imaging utility offers novel opportunities (the high sensitivity and spatial resolution of optical imaging ex vivo with the high spatial resolution in vivo and anatomical information provided by MRI) and serves as a control as well, where MR data can be co-registered and spatially colocalized with confocal microscopy data. The use of these bimodal MLs further allowed to evaluate the in vivo biodistribution and pharmacokinetics of the particles and the effect of PEGylation thereon (plasmatic half-lives of 70 min and 12.5 h for non-PEGylated and PEGylated MLs, respectively).37 In vitro, the addition of fluorescent probes to the MLs enabled to evaluate, in detail, the effect of magnetic targeting of these particles on the uptake by PC3 human prostatic adenocarcinoma cells (see Figure 2).38 The correlation of iron oxide and the fluorescent probe furthermore allows to assess the intact structure of the MLs before, during, and after cell internalization.38

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siRNA.39 MLs therefore represent an interesting delivery vehicle which can be employed for the magnetically targeted local delivery of drugs furthermore enabling real-time and in vivo MR assessment of the targeting efficiency. Using 5-fluorouracil and SPIO in one large liposome, released cargo molecules can be successfully demarcated from intact MLs by means of a recently developed MRI/MRS technique.43 Intact MLs showed broadening of the 19 F 5-fluorouracil resonance line as line width is inversely proportional to T2 *, but free fluorouracil appears as a narrow resonance line. Furthermore, the use of 19 F-MRI allows for a quantitative correlation between the MRI signal and number of MLs and exhibits no tissue background signal.19,20 These studies highlight the great potential role of MLs and MRI in general to optimize drug delivery strategies.

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DYNAMIC MRI OF LOW IRONCONTAINING LARGE MLS FIGURE 2 | Confocal microscopy images of PC3 cells incubated with polyethylene glycolylated rhodamine-labeled large magnetoliposomes (MLs) (400 nmol of total lipids per 106 cells; [lipids] = 0.4 mM) in the presence of a 0.4 T magnet attached under the coverslip. Magnetic influence domain (a). Cell cytoplasmic membrane labeled by fluorescent linker FITC-PKH67 was seen in green while rhodamine-MLs are seen in red. Images viewed from the top cell surface (921 × 921 µm) of rhodamine fluorescence (b), FITC fluorescence (c), and superimposition of both (d). White bar represents 230 µm. (Reprinted with permission from Ref 38. Copyright 2008 Elsevier.)

LARGE MLS AS THERANOSTICS Employing MLs instead of common liposomes for drug delivery purposes enables following the in vivo distribution of the MLs and assessing the efficacy of their targeting.39 The presence of the iron oxide cores can further be exploited by applying an alternating magnetic field, which will lead to a heating effect which can be used for magnetic cancer hyperthermia in conjunction with the drugs.40 Using specific lipid compositions for the MLs, so-called thermosensitive liposomes can be created, which will turn from a solid-like phase to an aqueous gel-like phase when reaching a critical temperature. Using carboxyfluorescein as a model compound, it was shown that application of an alternating magnetic field augmented its release both in phantom studies as well as in vivo in rats.41,42 Fluorescent, cationic liposomes encapsulating both Feridex and cyclooxygenase-2 siRNA revealed potent and specific downregulation in vitro. In vivo, the multimodality allowed to evaluate the biodistribution both by MRI as well as fluorescence microscopy, showing an intratumoral delivery of the Vo lu me 3, March/April 2011

While the confinement of (U)SPIOs in vesicles enhances T2 contrast, the use of very low levels of (U)SPIOs predominantly enhances T1 contrast.44 Therefore, it is important to be able to control and evaluate the number of iron oxide NPs within a liposome, for instance by a magneto-optical methodology which was suggested by Cintra et al.45 Alternatively, the iron content in liposomes can also be determined using spetrophotometric measurements using Tiron,46 inductively coupled plasma-mass spectrometry,47 magnetophoresis, or electronic spin resonance.48 By carefully tailoring the incorporation of low levels of citrate-stabilized USPIOs and by incorporating a diethylene triamine pentaacetic acid (DTPA)–lipid conjugate into the ML bilayer which could be used to bind Gd3+ , the T1 /T2 ratio could easily be modulated and optimized.49,50 These particles had an r1 /r2 ratio of 0.633 at 3 T compared to a 0.032 ratio for Resovist, highlighting the efficient longitudinal relaxation properties.51 When these particles gradually accumulated within a specific region, the augmented concentration of USPIOs could be monitored by dynamic MRI and was shown to allow visualization of peritumoral vasculature by means of ring enhancement surrounding colorectal liver metastases.51 These initial studies require further optimization of the ML constitution and the ratio of Gd3+ and iron oxide. In an ideal situation, the presence of Gd3+ ions would augment the T1 effect of MLs in the initial phase, where due to dilution in the blood, the initial iron concentration will also be very low and thus lead to predominantly T1 based effects. In the case of hepatic tumors, this results in a ring enhancement of the vasculature surrounding

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FIGURE 3 | (a–f) Electron micrographs of crystal morphologies and intracellular organization of magnetosomes found in various magnetotactic bacteria. Shapes of magnetic crystals include cubo-octahedral (a), elongated hexagonal prismatic (b, d–f), and bullet-shaped morphologies (c). The particles are arranged in one (a–c), two (e), or multiple chains (d) or irregularly (f). Bar equivalent to 100 nm. (Reprinted with permission from Ref 53. Copyright 1999 Springer-Verlag.)

the tumor mass. Upon accumulation of the MLs within the tumor mass, the augmented iron concentration will render the T2 -based effects more dominant, resulting in a darkening of the tumor mass. By dynamic MRI, this would allow to nicely outline the tumor mass itself at later time points, where, initially, the tumor vasculature can be visualized.

BIOGENIC MLS In nature, magnetotactic bacteria such as the Magnetospirillum species are a heterogeneous group of prokaryotes which are ubiquitous in aquatic environments and are characterized by the presence of unique intracellular magnetic structures known as magnetosomes.52 The iron mineral particles mostly consist of magnetite. They are characterized by a 202

narrow size distribution, uniform, species-specific crystal properties, and are enveloped by a lipid membrane (which also contains several proteins) (also see Figure 3).53 Due to the narrow size distribution of the iron oxide nanocores and the species-specific membrane, magnetosomes have attracted a lot of interest as MRI contrast agents. For MRI, it was shown that magnetosomes are expedient alternatives to synthetic ferrofluids and lead to a low detection threshold.54 Furthermore, similar to large MLs, fluorescently tagged magnetosomes have also been produced, allowing the bimodal detection of labeled macrophages to visualize inflammation.55 Recently, the high biocompatibility of bacterial magnetosomes has been shown by Sun et al.56 who found an LD50 of 62.7 mg/kg body weight of magnetosomes injected in the sublingual vein of rats. At 40 mg/kg body weight,

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no significant effects could be noted in terms of routine blood exam results, and liver and kidney function and magnetosomes did not cause an immune response or inflammation or alterations in the stimulation index of lymph cells. In the literature, it has already been shown that several attenuated bacterial strains show great potential in cancer therapy as these NP-loaded bacteria show a high and specific accumulation in several tumor types.57 In a similar manner, Wilhelm et al.58 recently demonstrated that the eukaryotic microorganism Dictyostelium discoideum was capable of internalizing (U)SPIOs, followed by the subsequent release of submicrometric vesicles loaded with the NPs (see Figure 4). Similar to large MLs, the confinement of the particles into vesicles resulted in an enhancement of the r2 /r1 ratio (which equaled 14), placing them among the best T2 -contrast agents as well.

incorporation of PEGylated lipids to increase blood circulation times or attach targeting entities. Large MLs, however, still possess an aqueous cavity, which allows to encapsulate hydrophilic drugs and they are also well suited for dynamic MRI, thanks to the controllable amount of iron oxide per particle. With regard to these properties, small MLs are less suitable, although the incorporation of hydrophobic drugs in the lipid bilayer remains possible, as has been shown for propranolol59,60 and 10-hydroxycampthothecin61 as model drug compounds. The use of small MLs as bimodal imaging agents or as theranostics therefore remains possible, but small MLs possess several unique properties which make them distinct from large MLs and other MR contrast agents and which will be discussed in the following section.

SPECIFIC PROPERTIES OF SMALL MLS SMALL MLS The interesting properties of large MLs are derived to a large extent from the flexibility of the liposomal coating, allowing easy surface manipulation. For small MLs, all these features are maintained, allowing the facile synthesis of bimodal contrast agents,25 (a)

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Small MLs consisting of 14-nm-diameter iron oxide cores onto which a lipid bilayer is immediately attached were first described in 1988 by De Cuyper and Joniau.16 For the production of MLs, a waterstable, lauric acid stabilized ferrofluid is dialyzed with a fivefold excess (in terms of phospholipid weight) of preformed small unilamellar vesicles. During the dialysis, the lauric acid is gradually replaced by the phospholipids as the phosphate moiety has a stronger affinity for the iron oxide surface than the carboxylgroup of lauric acid,62 leading to a strongly chemisorbed inner leaflet of the lipid bilayer. The outer leaflet is then formed according to a Langmuir adsorption model.26 As a result, the inner leaflet of the lipid bilayer resists extraction in detergents16 and organic solvent,63 whereas the outer leaflet of the lipid bilayer can easily be exchanged with lipids from other vesicles upon incubation.26 The latter procedure entailing a two-step protocol is frequently used for the production of cationic MLs, where neutral MLs will be incubated with cationic lipid-containing vesicles.64 This leads to an exchange of lipids from the outer leaflets of the ML and vesicle bilayers, resulting in an equilibrium when, under equimolar lipid concentrations, one third of the total amount of lipids from the vesicles have been incorporated in the ML bilayer.27 For more information on the specific properties of MLs and the process of lipid transfer, the interested readers are referred to Soenen et al.27 For MRI, the small size of the MLs and the lipid coating soon proved to offer many interesting applications. At a clinical magnetic field strength (1.5 T), the r2 /r1 ratio of 80 was unusually high as most USPIOs have r2 /r1 ratios of approximately 3.65 This unusually high value is thought to be caused by the phospholipid bilayer, which acts as a barrier

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for free water diffusion and reduces the relaxation of inner sphere water molecules. PEGylated MLs were shown to have excellent properties for bone-marrowtargeting MR contrast agents (see Figure 5).66 Using small MLs for direct stereotactic injection of contrast agent in the subventricular zone of the mouse brain in order to label endogenous neural progenitors in situ and track their migration along the rostral migratory stream (RMS) toward the olfactory bulb revealed that the small size of the particles led to background migration of MLs along white matter tracts, independent of any actual migration of neural progenitor cells.67 This study indicated that small (U)SPIOs are intrinsically able to visualize anatomic details (RMS) but they are not well suited for the in situ labeling of neural progenitor cells as noninternalized particles can migrate on their own and cover large distances in the white matter tracts.

found to be nontoxic and allowed excellent internalization in a wide variety of cell types tested, including primary human umbilical vein endothelial cells,70 murine C17.2 neural progenitor cells,71 PC12 rat pheochromocytoma cells, and mesenchymal stem cells (MSC, see Figure 6).72 Replacing DSTAP by an inhouse produced DPPE-succinyl-tetralysine conjugate could further enhance cell internalization, leading to 95 ± 6 pg Fe per cell in 3T3 fibroblasts after 24 h incubation at 100 µg Fe/mL.73 Although no direct effects on cell viability could be noted, these particles revealed that when the amount of intracellular iron oxide NPs raises too high, cell physiology is greatly affected as evidenced by a diminished cell spreading and impeded proliferation. Subsequently, it was discovered that this is also true for all common iron oxide NPs [Resovist, Endorem (Feridex in the US)] when they reach high intracellular levels.71 Although high intracellular iron oxide NP levels are beneficial to enhance MR contrast and to allow long-term follow-up of dividing cells, it seems that the amount of NPs which can be safely internalized is limited. Using well-tolerated concentrations of cationic MLs and Endorem for labeling MSCs showed superior imaging properties of MLs for in vivo cell visualization in the mouse brain (see Figure 6). From these data it can be noted that MLs are more sensitive than Endorem if comparable iron concentrations are used. Besides the excellent uptake properties of the cationic MLs, the higher sensitivity may be explained by intracellular aggregation (see below) which results in stronger T2 /T2 * effects compared to when similar intracellular iron concentrations are achieved. When particles only were injected, migration of the particles along the corpus callosum is comparable. But for labeled cells which remain near the injection site, the blooming effect for ML-labeled cells results in a lower detection limit, which creates the potential to detect even limited numbers of cells in vivo.

CATIONIC MLS ENABLING HIGH CELLULAR UPTAKE

THE EFFECTS OF INTRACELLULAR NP DEGRADATION ON MR SIGNAL

The incorporation of cationic lipids in the outer leaflet of the ML bilayer greatly enhanced cellular uptake of the particles.68 Careful attention has to be paid to potential toxic effects,46 however, as cationic lipids intrinsically exert cytotoxicity.27,69 Thanks to the reproducible and controllable manipulation of the ML production, different cationic lipid types and concentrations could be tested, finally putting forward 3.33% DSTAP MLs as the ideal MLs for cell labeling, resulting in an unusually high concentration of 47 ± 7 pg Fe/cell for 3T3 fibroblasts after 4 h of labeling with 100 µg Fe/mL medium.46 These particles were

In view of MRI analysis of labeled cells, one important issue is the intracellular degradation of the contrast agent. Previously, it was shown that Sinerem was completely degraded when present in the lysosomal environment of macrophages after 7 days.74 Arbab et al.75 further showed that pH was an important factor in degradation of Endorem, revealing a significant decrease in T2 relaxation rates with time when Endorem was exposed to pH 4.5. Recently, we showed that this degradation also occurred with Resovist, but was far more pronounced for VSOP particles and far less for lipid-coated MLs (see Figure 7).72

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FIGURE 5 | Coronal FSE 3000/45 millisecond images before (a, c) and 1 h after (b, d) injection of polyethylene glycolylated small magnetoliposomes (MLs), obtained in 6–8-week-old rats. Arrows outline bone marrow uptake. (Reprinted with permission from Ref 66. Copyright 1999 Wiley-Liss.)

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FIGURE 6 | Cell uptake and magnetic resonance (MR) contrast generation by magnetoliposomes evaluated. (a) Magnetoliposome uptake by stem cells and MR contrast generation were evaluated and compared to Endorem by incubating mouse mesenchymal stem cells (mMSCs) for 24 h with magnetoliposomes (MLs) or Endorem suspensions at different Fe concentrations (50, 100, 200, and 0 µg Fe/mL as indicated on the figures). One lakh mMSCs were harvested, washed, suspended in 1.5% agarose, and collected in 0.2 mL microcentrifuge tubes, held together in a plastic container containing 1.5% agarose. Three-dimensional T2 *-weighted MR images (panel a, first row; TR/TE/θ = 200 milliseconds/15 milliseconds/27◦ , 234 µm3 isotropic resolution) and quantitative T2 -maps (6 slices of 0.35 mm, TR = 3000 milliseconds, 16 echoes, TE = 10.098 milliseconds, spatial resolution = 215 µm) were acquired on a Bruker 9.4 T small animal scanner. Relative T2 -values were 0.67 (50 µg Fe/mL), 0.64 (100 µg Fe/mL), and 0.53 (200 µg Fe/mL) for ML-labeled stem cells, and 0.85 (50 µg Fe/mL) and 0.53 (200 µg Fe/mL) for Endorem-labeled cells, relative to unlabeled cells (T2 = 1.00). (b) To evaluate in vivo the ML-mediated MR contrast distribution and contrast generation compared to Endorem, mice (C57Bl/6, n = 2) were stereotactically injected in the striatum with MLs (panel b, first row) or Endorem (panel b, second row) (2.5 µg of total Fe in 3 µL). Representative 3D T2 *-weighted MR images (TR/TE/θ = 100 milliseconds/12 milliseconds/20◦ ) at 1, 3, and 4 weeks post-injection are shown. After the last time point, mice were sacrificed and processed for histology. The fourth image shows light microscopy photographs of Prussian Blue staining at the vibratome section (50 µm) corresponding to the MR image, visualizing the distribution of Fe(III) around the injection site (scalebars = 1000 µm). The inset shows the site of injection at a higher magnification (scalebar = 200 µm). (c) MLs were evaluated as an MR contrast agent for cell labeling, compared to Endorem. mMSCs were labeled in cell culture with optimized concentrations of MLs (100 µg Fe/mL) and Endorem (250 µg Fe/mL). Subsequently, the labeled stem cells were stereotactically injected in mouse striatum (C57Bl/6, n = 2, 10,000 cells in 3 µL). Mice were MR imaged at 1, 3, and 4 weeks post-injection with the same scanning parameter set. The first three images show representative 3D T2 * MR images of the three consecutive time points. Prussian Blue staining reveals the distribution of Fe(III)-labeled stem cells.

These data highlight the importance of the coating of the iron oxide NPs on resisting endosomal degradation. It also indicates that small MLs are more resistant to such degradation compared to citrate-or Vo lu me 3, March/April 2011

dextran-coated particles due to the strong attachment of the inner leaflet of the lipid bilayer. Next to having grave effects on the MR properties, degradation of the NPs also has pronounced effects on cell functionality,

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time for the four NPs (MLs, Endorem, VSOP, and Resovist) incubated at 200 µg Fe/mL in 20 mM sodium citrate containing cell culture medium at different pH (a: pH 7.0; b: pH 5.5; c: pH 4.5; n = 4). (d–g) Representative T2 * maps obtained for the various particles (d: ML; e: Endorem; f: VSOP; g: Resovist) in the above-described medium at pH 4.5. The samples were collected at different time points after addition of the NPs to the acidic culture medium and are represented clockwise in terms of increasing incubation times, going from (a): pure agar to 12 h; 24 h; 48 h; 72 h; 1 week, and 2 weeks. (h–k) T2 * values obtained when calculating the respective T2 * maps of the four NPs (h: ML; i: Endorem; j: VSOP; k: Resovist) at pH 7.0, 5.5, and 4.5 as a function of different incubation times. Significant increases of T2 * relaxation times of NPs treated at pH 5.5 or pH 4.5 compared with the values obtained at pH 7.0 are indicated (*P < 0.05; **P < 0.01; ***P < 0.001). (Reprinted with permission from Ref 72. Copyright 2010 Wiley-VCH.)

as was observed when using the PC12 cell model system.72 Although the slow intracellular degradation and high persistence of small MLs enable efficient long-term follow-up of labeled cells, these features may also pose a limitation to direct in vivo applications of these particles. Upon intravenous administration, the particles will end up in the liver following clearance by the reticuloendothelial system and will accumulate there in high concentrations. For most iron oxide NPs, their rapid clearance through degradation or along the hepatobiliary route or by renal clearance diminishes the possible induction of secondary toxic effects. For MLs, the long persistence of high doses of MLs in the hepatic system may impede correct liver functioning in the long term. More studies are needed to investigate this predicament and to compare small MLs with other iron oxide NPs in terms of efficiency of clearance via the hepatobiliary route or renal clearance and possibly associated impediments of organ function. 206

INTRACELLULAR CLUSTERING OF SMALL MLS The high resistance of small MLs to pH-dependent degradation and the minimal effects on cell functionality compared with VSOP, Endorem, and Resovist suggested that small MLs are relatively stable in intracellular compartments. Typically, lipid structures are broken down by phospholipases (PLs), such as PLA1 or PLA2 which cleave the fatty acyl chains bound to the first or second carbon atom from the glycerol group, respectively.76 In an in vitro setup, the effect of purified PLA2 on the ML coating was investigated, showing a clear degradation of the outer lipid layer, whereas the inner lipid layer remained intact.72 This resulted in monolayer coated iron oxide particles which have a hydrophobic surface and will cluster in an aqueous environment (see Figure 8). The clustering of the particles enhances their hydrodynamic diameter

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FIGURE 8 | Clustering of magnetoliposomes. (a–e) Effect of PLA2 activity on MLs as indicated by (a) a decrease in pH of PLA2 -treated samples (gray) compared with control samples (black). (b) The ratio of phosphate over magnetite for PLA2 -treated samples (gray) and control samples (black). (c) Hydrodynamic diameters of PLA2 -treated samples (gray) and control samples (black). (d) Optical micrograph of control particles and PLA2 -treated particles upon exposure to a 1-T magnetic field for 30 min. (e) T2 * relaxation times for PLA2 -treated samples (gray) and control samples (black) indicating the enhanced effect of induced clustering of the MLs on MR contrast generation. All data are expressed as mean ± standard deviation (n = 3). When appropriate, the degree of significance is indicated (*P < 0.05; **P < 0.01; ***P < 0.001). (f) Representative optical micrographs of C17.2 NPCs labeled with Endorem (top row) or MLs (bottom row) during 24 h, finally reaching similar average intracellular iron content. Media were removed and cells were kept in culture for the duration indicated. Cells were stained for iron oxide using DAB-enhanced Prussian Blue reagent and imaged 24 h (left column), 1 week (middle column), and 2 weeks (right column) post-nanoparticle incubation. Scale bars: 75 µm. The inset is an enlarged view of a single cell displayed in the main image. (Reprinted with permission from Ref 72. Copyright 2010 Wiley-VCH.)

and their T2 contrast and results in an asymmetric distribution of the amount of iron upon cell division. This intracellular clustering of the MLs has significant effects on the MRI detectability and the labeling efficiency of rapidly proliferating cells, such as C17.2 neural progenitor cells. Upon cell division, some cells will contain sufficient iron for cell detection by MRI because of the intracellular clustering of MLs and the asymmetric distribution of the clustered particles. As can be seen from Figure 8(f), when cells were labeled with Endorem or MLs, reaching identical intracellular iron levels, Endorem-labeled cells showed less Vo lu me 3, March/April 2011

Prussian Blue-positive NPs than ML-treated cells after 1 week. After 2 weeks, no more Endorem particles could be detected, whereas large ML clusters could still be observed. This highlights the high stability of the MLs against intracellular degradation and makes these particles more suited for long-term detection of labeled cells, even upon recurrent cell divisions.25,70

CONCLUSION Biomedical imaging research is currently progressing toward the development and use of bimodal imaging

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agents or theranostic materials, where targeting entities, imaging modalities, and therapeutics agents are all present in one single nanobiosystem. In this regard, MLs offer an interesting platform, where small iron oxide nanocores and other hydrophilic imaging or therapeutic agents can be combined in the aqueous central cavity of liposomes (large MLs) or for hydrophobic compounds, to be incorporated in the lipid bilayer of the MLs (both large and small MLs). The high flexibility and ease of preparation enables to carefully fine-tune the composition of the liposomes, enabling to incorporate fluorescent–lipid conjugates, cationic or functionalized (with PEG, antibodies, or peptides) lipids separately or all combined into one single ML. These features bestow the MLs with a high cellular uptake efficiency compared to dextran-coated iron oxide NPs. Special types of large MLs are the bacteria-derived MLs, including magnetosomes from magnetotactic bacteria with a very narrow size distribution and many possible shapes of the iron oxide cores or exosomes from species which were previously labeled with iron oxide NPs. These particles offer interesting potential to be used in cancer treatment and imaging, and more research is required to

explore the efficacy of these particles in MR contrast generation and their tumor-tracking abilities. In terms of MR properties, small MLs appear to be rather resistant to pH-dependent degradation in contrast to citrate- or dextran-coated particles. This is due to the high stability and strong binding of the inner layer of the lipid bilayer to the iron oxide core, which efficiently shields the iron oxide core from degradation. Furthermore, after cellular internalization, the outer lipid layer of the MLs can easily be degraded, leaving the inner layer exposed, rendering the MLs with a hydrophobic surface. This results in an intracellular aggregation of the MLs, with an enhanced transversal relaxation as a result of the clustered iron oxide cores. These features render small MLs highly useful for long-term imaging of labeled cells. The extent of the intracellular aggregation and the effect thereof on MR contrast generation in labeled cells requires more in-depth investigation. Also, long-term studies using ML-labeled stem cells which are stereotactically injected would be highly useful and might open a new window for efficient cell tracking studies in the long term.

ACKNOWLEDGEMENTS SJS is a post-doctoral fellow from the FWO Vlaanderen. MDC and UH are recipients of an IWT grant sponsoring project SBO80017 and received financial support from the KULeuven Center of Excellence IMIR. UH received financial support from the European Commission for EC-FP7 network ENCITE (2008-201842).

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FURTHER READING Aime S, Carrera C, Delli Castelli D, Geninatti Crich S, Terreno E. Highly sensitive MRI chemical exchange saturation transfer agents using liposomes. Angew Chem Int Ed 2005, 44:1813–1815. An interesting review on MR methods for molecular imaging is given by: Vande Velde G, Baekelandt V, Dresselaers T, Himmelreich U. Magnetic resonance imaging and spectroscopy methods for molecular imaging. Q J Nucl Med Mol Imaging 2009, 53:565–585. A technical note on how to assess the effect of iron oxide nanoparticles on cell physiology, viability, and functionality is given by: Soenen SJ, De Cuyper M. How to assess cytotoxicity of (iron-oxide-based) nanoparticles. A technical note using cationic magnetoliposomes. Contrast Media Mol Imaging 2010, doi: 10.1002/cmmi390. Cohen B, Ziv K, Placks V, Harmelin A, Neeman M. Ferritin nanoparticles as magnetic resonance reporter gene. Wiley Interdisc. Rev. Nanomed. Nanobiotechnol 2009, 1:181–188. Koole R, Mulder WJ, van Schooneveld MM, Strijkers GJ, Meijerinck A, Nicolay K. Magnetic quantum dots for multomodal imaging. Wiley Interdisc. Rev. Nanomed. Nanobiotechnol 2009, 1:475–491.

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Magnetoliposomes as magnetic resonance imaging contrast agents.

Among the wide variety in iron oxide nanoparticles which are routinely used as magnetic resonance imaging (MRI) contrast agents, magnetoliposomes (MLs...
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