Article pubs.acs.org/ac
Giant Vesicles Containing Superparamagnetic Iron Oxide as Biodegradable Cell-Tracking MRI Probes Taro Toyota,†,∥ Naoto Ohguri,† Kouichi Maruyama,‡ Masanori Fujinami,† Tsuneo Saga,§ and Ichio Aoki*,§ †
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi, Inage, Chiba, Chiba 263-8522, Japan ‡ Research Center for Radiation Protection and §Molecular Imaging Center, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage, Chiba, Chiba 263-8555, Japan S Supporting Information *
ABSTRACT: A major breakthrough in in vivo cellular imaging has been the clinical/preclinical use of magnetic resonance imaging (MRI) with contrast agent. Superparamagnetic iron oxide (SPIO) is a promising candidate for the development of smart MRI probes for cell-tracking. In the present study, we describe biodegradable probes made of giant vesicles (GVs; closed lipid membranes with diameters >1 μm) that encapsulate SPIO for use as an MRI contrast agent. These SPIO-containing GVs (SPIO-GVs) exhibited excellent contrast enhancement in the single cell of medaka fish (Oryzias latipes) embryos immediately after their microinjection, and this enhancement disappeared when the GV membranes were destroyed. Our results demonstrate that SPIO-GVs are useful MRI probes for single cell-tracking that have minimum cytotoxicity and will greatly improve clinical/preclinical in vivo cellular imaging techniques.
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limited to experimental studies. In contrast to the use of large SPIO particles, the use of smaller SPIO particles, especially of the nanometer-sized SPIO particles that are currently used for cell tracking, results in loss of signal detectability, as well as accumulation of the particles to a high density within cells, which may also be toxic to the cells. These issues must be overcome before cell-tracking applications that are safe and low toxicity can be realized. Vesicles with phospholipid membranes that encapsulate SPIO at a high number density will provide the solution to the above problems. Phospholipid membranes are natural membranes that are degradable in vivo. Encapsulation of SPIO at high number density into giant vesicles (GVs; diameter, >1 μm) provides SPIO-containing GVs (SPIO-GVs) that can be used as highly sensitive MRI probes, and their MRI enhancement effects can be “switched off” by means of various vesicle-destroying stimuli such as changes in pH, temperature, ionic strength, or enzyme activity.20−25 However, conventional GV preparation methods such as film swelling26 or electroformation27 cannot be used to encapsulate a high number density of SPIO particles inside GVs. This is because such methods are based on the self-organization of lamellae for the formation of SPIO-GVs, which results in nonuniform GVs in which the number of lamellae is affected by the number density of SPIO inside of the GV under physiological conditions.28
t is necessary to develop new technologies for imaging the living body using cell-tracking systems in order to ensure good spatial resolution, sensitivity, and detectability. High-field magnetic resonance imaging (MRI) with contrast media is already in clinical use, and the design of new MRI probes for pharmaceutical and medical applications has attracted much attention.1−6 Superparamagnetic iron oxide (SPIO) is a promising candidate for the development of smart and functional probes for cell-tracking systems.6−15 The viability of cells or living organs following introduction of SPIO nanoparticles coated with carboxydextran (known as ferucarbotran) for clinical purposes has already been examined in other studies.6,9,12−16 Some of these reports indicated that SPIO nanoparticles in the cytosol are dissolved in lysosomes or are captured by macrophages outside of the cells.6,12−15 Because MRI contrast enhancement with SPIO depends on the size or number density of SPIO particles that are condensed into a certain space, the conventional uses of SPIO have focused on the accumulation of SPIO into cells or organs.6,9,11,16−18 The use of larger SPIO particles, such as micrometer-sized particles, allows detection of single cells in a rat embryo using 11.7-T MRI.19 However, several issues remain to be resolved before single-cell MRI tracking techniques can be applied safely in the clinical/preclinical trials.. Thus, although micrometer-sized particles can provide excellent detectability owing to the strong susceptibility effect, larger SPIO particles (>1 μm) are not degraded but remain permanently in the cells and may induce long-term toxicity or change the function of the labeled cells. Therefore, analysis of micrometer-sized SPIO particles is © 2012 American Chemical Society
Received: November 25, 2011 Accepted: April 2, 2012 Published: April 2, 2012 3952
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precipitated SPIO-GVs were collected through a hole opened at the bottom of the centrifugation tube. Microinjection of Medaka Embryos. Medaka (drR strain) were maintained under breeding conditions: a 14 h light/10 h dark photoperiod at about 26 °C. Fertilized eggs were removed from the attaching filament and incubated until they reached the 4-cell stage. The SPIO-GVs dispersion or normal saline solution (approximately 0.5 nL) was injected into one cell of the 4-cell stage embryo with a micropipet (i.d. < 10 μm) made from a sterilized, siliconized, fiber-filled glass capillary tube (1 × 90 mm, GD-1, Narishige, Tokyo, Japan) by means of a vertical puller (PB-7, Narishige). DIC Microscopy and Fluorescence Microscopy. Differential interference contrast (DIC) micrographs of SPIO-GVs and medaka embryos were acquired with an Olympus IX71 microscope (Tokyo, Japan) equipped with a halogen lamp for fluorescence microscopy. A confocal laser scanning fluorescence micrograph of SPIO-GVs stained with 4-(4(dihexadecylamino)styryl)-N-methylpyridinium iodide was obtained with an Olympus FV1000 microscope system with a 458-nm semiconductor laser and a detection filter (505−605 nm). MRI. For in vitro MR imaging of SPIO-GVs, we adjusted the numerical density of SPIO-GVs to 6 × 103 mL−1 with a counting cytometer and then introduced the dispersion into a handmade polymethylmethacrylate well made of two concentric circular walls (i.d. = 10 and 18 mm; height = 10 mm). The dispersion was mixed with a 1.5% (w/w) CaCl2 solution and then a 12% (w/w) pectin solution, and the well was sealed for 3 h at 22−25 °C. For MR imaging, the medaka embryos were fixed with 4% (w/w) paraformaldehyde aqueous solution for 5 h. Each fixed embryo was placed on a silicon sample plate (one spot on a 7-spot well, i.d. = 5 mm, height = 5 mm, Dai Nippon Printing, Tokyo, Japan), and a 6% (w/w) pectin solution and a 1.5% (w/w) CaCl2 solution were added to the embryo. The MR images were acquired in a 7.0-T, 400-mm horizontal bore magnet (Kobelco and Jastec, Tokyo, Japan) interfaced to a Bruker Avance-I console (Bruker Biospin, Tokyo, Japan). For embryo measurements, a 72-mm-diameter volume coil was used for transmission (Bruker Japan, Tokyo, Japan) and a 2-ch phased array surface coil was used for reception (Rapid Biomedical, Rimpar, Germany). The silicon sample plate was placed in a handmade sample holder for detection using the surface coil. Pure water was prepared in one spot of the sample plate for a reference MR signal (see Supporting Information Figure S1). The sample was maintained at approximately 23.0 ± 0.5 °C (room temperature). The measurements were performed as follows: three-dimensional T1-weighted imaging with a rapid acquisition of relaxation enhancement (RARE) sequence with a fat suppression preparation pulse; repetition time = 350 ms; echo time = 18.6 ms; matrix size = 256 × 224 × 128; field of view = 15.44 mm × 13.44 mm × 7.68 mm; RARE factor = 4; and number of acquisitions = 8. The nominal voxel resolution was 60 μm × 60 μm × 60 μm. The total acquisition time was 5 h and 34 min. Two-dimensional T1-weighted incoherent gradient-echo imaging was carried out with a fast low angle shot sequence with a fat suppression preparation pulse; repetition time = 300 ms; echo time = 5.5 ms; flip angle = 60°; matrix size = 512 × 512; field of view = 36 mm × 36 mm; slice thickness = 0.5 mm; slice gap = 0.5 mm; number of slices = 3; and number of acquisitions = 48. The nominal voxel resolution was 70 μm × 70 μm × 500 μm. The total acquisition time was 2 h and 3 min. Image reconstruction and analysis were
Pautot et al. have reported a water-in-oil (W/O) emulsion centrifugation method for the preparation of GVs29 and a flow cytometric study by Nishimura et al. demonstrated that this method affords GVs whose vesicle membranes are close to being a single lamella.30 Moreover, this technology allows encapsulation of a high density of SPIO because the water phase inside the W/O emulsion droplets within which SPIO particles are dispersed remains isolated from the outer water phase as the droplets pass through the water−oil interface under the influence of centrifugation, which results in the formation of SPIO-GVs. Thus, the main advantage of this SPIO-GV compared to previously developed probes is that it is a probe that can be used for single cell tracking using MRI. Medaka fish (Oryzias latipes) are of increasing interest as a model vertebrate in developmental and evolutionary biological studies. In addition to features that medaka share in common with zebrafish, medaka have several further advantages including a small genome and availability of inbred strains and various mutants. In terms of bioimaging studies, medaka have the advantages of having a large and transparent embryo and of undergoing in vitro fertilization.31 We therefore used medaka for the present study since their use made it possible to compare fluorescence micrographs and MR images of injected SPIO-GVs marked by a fluorescent lipid, in order to determine the usefulness of SPIO-GVs for cell-tracking studies. Our goal in the current study was to construct a GV-based biodegradable MRI cell-tracking probe that would allow high detectability during the tracking period and safe excretion after the observation period. We demonstrate the MRI contrast capability of the SPIO-GVs in a single cell in fertilized eggs of the medaka fish and show that this contrast is disappeared when the GV membranes are destroyed.
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MATERIALS AND METHODS Reagents. Egg lecithin, cholesterol, glucose, sucrose, 2amino-2-hydroxymethyl-1,3-propanediol (Tris), glycine, and sodium acetate were purchased from Kanto Chemical Co. (Tokyo, Japan). SPIO suspension (final iron concentration, 40 mg mL−1; average particle diameter, 93 nm) containing carboxydextran (MW = 20 kDa, 50 mg mL−1) was supplied as ferucarbotran by Meito Sangyo Co., Ltd. (Nagoya, Japan). Dimyristoyl-phosphatidyl glycerol (DMPG) sodium salt, liquid paraffin (specific density; 0.86−0.89 g mL−1 at 20 °C) and calcium chloride were purchased from Wako Pure Chemical Industries, Inc. (Tokyo, Japan). 4-(4-(Dihexadecylamino)styryl)-N-methylpyridinium iodide and pectin were obtained from Invitrogen (Carlsbad, CA) and Sigma-Aldrich (St. Louis, MO), respectively. These reagents were used without further purification after purchase. All lipids were stored at −20 °C under nitrogen. Water was distilled and then subjected to ion exchange with a Milli-Q water system (Millipore; Bedford, MA). Preparation of SPIO-GVs. The SPIO suspension (final iron concentration, 40 mg mL−1; average particle diameter, 93 nm) containing carboxydextran was mixed into a buffered saline solution of 100 mM Tris-HCl (pH 7.8) and 100 mM NaCl. After the SPIO suspension (30 μL) was dispersed in liquid paraffin (300 μL) containing egg lecithin (9 mM), DMPG (1 mM), and cholesterol (1 mM), the resulting water-in-oil (W/ O) emulsion (400 μL) was layered on a buffered saline solution (1 mL) of 100 mM Tris-HCl (pH 7.8), 100 mM NaCl, and sucrose. After incubation at 4 °C for 10 min, the W/O emulsion was centrifuged at 18 800g at 20 °C for 30 min. The 3953
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Figure 1. (A) Differential interference contrast micrograph of SPIO-GVs. The SPIO-GVs appear as dark brown particles because SPIO dispersions scatters visible light. (B) Fluorescence micrograph of typical SPIO-GVs labeled with a lipid membrane fluorescent dye (excitation with a 458-nm semiconductor laser; detection with a 505- to 605-nm bandpass filter). The micrograph was obtained using a confocal scanning laser fluorescence microscope. Scale bars = 50 μm. (C) In vitro MR image (gradient echo, 1.5 mm × 1.5 mm) of SPIO-GVs in a pectin−calcium gel sample in one chamber of the apparatus. A DIC micrograph of an SPIO-GV in the boxed area of this sample is shown below.
Figure 2. Differential interference contrast micrographs and MR images (gradient echo with fat suppression) of fixed medaka embryos at the 4-cell stage. (A−C) The black arrows indicate (A) untreated cells, (B) cells microinjected with approximately 0.5 nL of the SPIO-GV dispersion, and (C) cells treated with the SPIO dispersion in which the GV membranes had been destroyed using a surfactant. Scale bars = 200 μm. As shown in panel A, the cells in blastomeres (a) of the untreated cells displayed positive contrast while oil droplets in the embryo (b) displayed negative contrast due to the chemical shift of the fat molecules. (D) Differential interference contrast and fluorescence micrographs of a medaka embryo in which some cells had been microinjected with a fluorescent SPIO-GV dispersion. A few fluorescent spots can be seen in the cells in the enlarged image of the boxed area (excitation filter, 460−480 nm; detection filter, >515 nm).
a culture chamber. Using a stereoscopic microscope, we carefully assessed the percentage of dead or abnormal embryos (Supporting Information Figure S2, the abnormality ratio; the number of abnormally developed or dead embryos divided by the total number of assessed embryos) based on the morphology of organ development, in particular on the formation of the notocord, heart, and head structure.32−34
performed by ParaVision (Version 4, Bruker Biospin) and MRVision (Version 1.5, MRVision Co., Winchester, MA). The accuracy and linearity of the gradient coil of the MRI system were routinely adjusted according to the manufacturer’s maintenance guidelines (Bruker Japan). Assessment of Medaka Embryos. During incubation at 28 °C for over 2 days, the injected medaka embryos were left in 3954
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To determine whether the SPIO-GVs can act as MRI contrast agents in living embryos, we acquired in vivo micro MR images of medaka embryos that were injected with SPIO-GVs. We first examined a noninjected embryo that had been fixed in paraformaldehyde and embedded in a pectin−calcium gel, using both DIC and MRI imaging. We observed no dark spots at the location of the blastomeres in this MR image (Figure 2A), whereas the oil droplets in the embryo did show up as dark spots in the MR image owing to the chemical shift of the fatty molecules.36 We next acquired an MR image of an embryo into which approximately 0.5 nL of the SPIO-GV dispersion (8 × 106 particles mL−1, counting using a cell counter) was injected into one cell of a 4-cell stage embryo with a glass capillary and the embryo was then fixed with paraformaldehyde. Dark spots with a typical diameter of 200 μm were observed at the location of the blastomeres in the MR images of these injected cells (Figure 2B). In contrast, cells injected with SPIOGVs that were destroyed by surfactant treatment showed no dark areas in the MRI (Figure 2C). The cellular distribution of the intact SPIO-GVs that was detected using MRI was consistent with the distribution of fluorescent-labeled SPIOGVs that was determined using fluorescence microscopy (Figure 2D). Note that only a few SPIO-GVs were detected in the blastomeres using fluorescence microscopy (Figure 2D). To assess the viability of the SPIO-GV-injected embryos, we carefully observed the development of medaka embryos after microinjection of the SPIO-GV dispersion and compared it to that of saline-injected embryos. There was no statistically significant difference between the viability of the medaka embryos after microinjection of the SPIO-GV dispersion and that of the embryos injected with the saline solution 6 or 48 h after microinjection (see Figure S2). Thus, we found that the abnormality ratio of the SPIO-GV-injected embryos was 5/18 (27.8%) while that of the saline injected embryos was 5/17 (29.4%) (Figure 3). This result provides further evidence that SPIO-GVs are useful as noninvasive MRI probes for cell tracking. We have shown above that the GV-based SPIO MRI contrast agent that we developed could enhance the MRI signal at a cellular level, that this enhancement could be eliminated after degradation of the SPIO-GV, and that these SPIO-GVs had low toxicity. Encapsulation of SPIO within GVs can therefore solve several previous problems associated with the use of iron oxidebased contrast agents: (1) although submicrometer range iron oxide particles have excellent detectability, they may not be excreted from the cells; and (2) if smaller iron oxide particles are used (10−100 nm), detectability may be lost or the particles may be toxic at higher concentrations. SPIO-GVs allow high detectability at a cellular level due to the susceptibility effect37,38 in addition to their biodegradability. Furthermore, signal enhancement by the SPIO-GVs can also depend on the process by which the membrane is degraded by enzymatic reaction such as lipases. Previous research regarding the development of SPIO-based MRI contrast agents has been focused on optimizing the diameters of such particles and on their surface modification.1−4,7,22,23,39−41 Although liposome-based SPIO particles have been reported,1−4,7,22,23 the purposes of these liposomeSPIOs were predominantly aimed at tissue targeting and/or attaining a longer half-life in the blood. Combining SPIO with GVs to enhance the detectability of the particles has not drawn much attention, not only because of the difficulty of encapsulating a high concentration of SPIO within GVs, but
Since the total number of assessed embryos was less than 20, a nonparametric analysis (Fischer’s exact test) was adopted for statistical analysis.
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RESULTS AND DISCUSSION To encapsulate a dispersion of SPIO particles (iron concentration, 40 mg mL−1; diameter, 93 nm) within GVs, we centrifuged an SPIO suspension dispersed in a lipidcontaining oil phase through an outer water phase that contained sucrose, sodium chloride, and Tris-buffer (see Materials and Methods). The concentration of sodium chloride and the pH of the Tris-buffer used corresponded to those of physiological conditions. The concentration of sucrose used was determined by assessment of differential interference contrast (DIC) microscopic images of the dispersion obtained after centrifugation (SPIO-GVs). Dispersed SPIO particles scatter visible light and therefore SPIO-GVs appear as dark brown particles in DIC images. When the outer water phase contained 0.5 M sucrose, the number density of SPIO-GVs reached a maximum (Figure 1A). When the sucrose concentration used was greater or less than 0.5 M, few SPIOGVs were formed following the W/O emulsion centrifugation, because the difference in osmolarity between the inner and outer sides of the vesicle membranes caused instability and rupture of the GVs (see Supporting Information Figure S3). By adding the membrane fluorescent dye, 4-(4(dihexadecylamino)styryl)-N-methylpyridinium iodide, to the W/O emulsion and subsequently analyzing the GVs present in the dispersion after centrifugation by means of confocal laser scanning fluorescence microscopy, we confirmed that the formed SPIO-GVs were composed of almost single lamellar membrane and had a diameter of 4.7 ± 2.2 μm (Figure 1B). The half-life of the obtained SPIO-GVs, which was determined by counting intact SPIO-GVs under the DIC microscope using a cell counter, was about 5 days at pH 7.7 at 20−23 °C. A similar half-life was observed in solutions buffered at various pH values in the range of 4.8−9.5 (Supporting Information Figure S4). The long half-life of the SPIO-GVs was probably due to the fact that phosphatidylcholine, the main membrane component of the SPIO-GVs, has a zwitterionic polar head that is charged at a wide range of pH values and to the fact that the high viscosity of the SPIO-GV dispersion (due to the presence of sucrose) kinetically stabilized the GV membranes and prevented aggregation.35 For in vitro 1H-MRI analysis of the SPIO-GVs, we mixed the SPIO-GV dispersion with a pectin−calcium ion gel and then filled a handmade concentric circular polymethylmethacrylate well with this mixture (see Materials and Methods). The gradient-echo MR image of the SPIO-GVs in the well showed dark spots that corresponded to the positions of SPIO-GVs as indicated by DIC microscopy (Figure 1C). To examine the capability of the SPIO that was condensed inside the GVs to function as an MRI contrast agent, we destroyed the membrane of the GVs by adding the surfactant sodium lauryl sulfonate to the SPIO-GV dispersion. Following confirmation that no GVs could be observed under the DIC microscope after this treatment, we then obtained an MR image of the dispersion in the gel sample. The disrupted SPIO-GVs in the sample clearly lost the capability to function as an MRI contrast agent. These data showed that a single SPIO-GV could be observed as a single particle by MRI imaging only as long as the MRI contrast agent, SPIO, was present at a high number density inside the GV. 3955
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This result will facilitate further development of cell tracking of multimodal probes using MRI for in vivo events such as development, tumor formation, or tissue reconstruction.
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CONCLUSION In conclusion, using the W/O emulsion centrifugation method, we developed GVs that encapsulate SPIO for use as a novel cell-tracking MRI contrast agent that is biodegradable in vivo. By using medaka embryos, we demonstrated that SPIO-GVs enhance MRI detectability and allow MR observations at the cellular level. The contrast enhancement of these SPIO-GVs was switched off when specific stimuli destroyed the membrane of the GVs through biodegradation. This ability to be switched off will be useful for application of these SPIO-GVs as a sensor that responds to external stimuli in addition to their application as a cell-tracking probe. We believe that our results will contribute to the provision of a noninvasive cell-tracking MRI system that will ultimately have clinical/preclinical applications.
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ASSOCIATED CONTENT
S Supporting Information *
Figure 3. Assessment of the viability of medaka embryos microinjected with the SPIO-GV dispersion. Damage to the embryos due to microinjection of the SPIO-GVs was minimal, and was similar to that observed at 6 and 48 h after injection with a saline solution (SS). There was no statistically significant difference (N.D.) between the viability of the medaka embryos after microinjection of the SPIO-GV dispersion and that of the embryos injected with the saline solution.
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-43-206-3272. Fax: +81-43-206-3276. E-mail: aoki@ nirs.go.jp.
also because of the instability of the GV membranes. Although conventional GV preparation methods28 can yield stable GVs even under physiological conditions, GVs with encapsulated SPIO nanoparticles get distorted in a high MR field due to the electromagnetic force.42−45 Here, we used the W/O emulsion centrifugation method to encapsulate SPIO within GVs.29,30 By adjusting the sucrose concentration of the solution through which the W/O emulsion was centrifuged, we were able to achieve a balance between the osmolarity of the solution outside of the GVs and the density difference between the inside and the outside of the GVs, resulting in successful SPIOGV formation. The formed SPIO-GVs in buffered solutions were relatively stable with a half-life of 5 days; it should be noted that these SPIO-GVs were stable during MRI detection in the presence of a high magnetic field. Thus, the SPIO was retained within the GV until the GV was biodegraded. The stability of the SPIO-GVs is probably due to the W/O emulsion centrifugation method used for their formation, which affords GVs with membranes that contain liquid paraffin, an oil-phase compound. Incorporation of liquid paraffin into the GV membrane can enhance SPIO-GV stability by inserting between the intramembrane phospholipids46 under a high magnetic field. Moreover, even though the diameter of the SPIO-GV that is shown in the DIC micrograph was 20 μm, the expanded diameter of the corresponding dark spot in the MR image was 200 μm due to the susceptibility effect. This effect results in good detectability of SPIO-GVs in cell-tracking using MRI. The SPIO-GVs demonstrated good detectability and spatial resolution even in the fertilized egg of medaka, which suggests that they will be useful for cell-tracking studies. Medaka has drawn much attention as an model animal for bioimaging, especially for fluorescence whole in vivo imaging that employs genetic engineering of the green fluorescent protein and its derivatives.47 Here, we found that MR images of medaka cells gave a homogeneous T1 contrast (except for the oil droplets).
Present Address ∥
Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully thank Shigeyoshi Saito and Misao Yoneyama (Molecular Imaging Center, National Institute of Radiological Sciences) for operation of the MRI instrument. We also thank Jeff Kershaw for helpful discussions. The analysis of giant vesicles by confocal scanning laser fluorescence microscopy was conducted with the help of the Association of Graduate Schools of Science and Technology, Chiba University. This work was partly supported by the Urakami Food and Food Culture Foundation, the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program), and by KAKENHI (JSPS).
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dx.doi.org/10.1021/ac2031354 | Anal. Chem. 2012, 84, 3952−3957
