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Gadolinium hexanedione nanoparticles for stem cell labeling and tracking via magnetic resonance imaging
Biomaterials 31 (2010) 5427e5435
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Gadolinium hexanedione nanoparticles for stem cell labeling and tracking via magnetic resonance imaging Ching-Li Tseng a, I.-Ling Shih b, Leszek Stobinski c, Feng-Huei Lin a, b, * a
Division of Medical Engineering Research, National Health Research Institutes, No.35, Keyan Rd, Zhunan Town, Miaoli County 350, Taiwan, ROC Institute of Biomedical Engineering, National Taiwan University, No.1, Sec. 1, Ren-ai Rd., Taipei City 100, Taiwan, ROC c Collage of Mechanical and Electrical Engineering, National Taipei University of Technology, Taipei, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2010 Accepted 19 March 2010 Available online 18 April 2010
The ability to trace transplanted stem cells and monitor their tissue biodistribution is prerequisite to an understanding of cellular migration after transplantation. Therefore, a new magnetic resonance imaging (MRI) contrast agent made of gadolinium hexanedione nanoparticles (GdH-NPs) was developed as a cell tracking agent. The GdH-NPs were fabricated by the microemulsion process. The physical characteristics, biocompatibility, and T1-MRI signal enhancement of these NPs were analyzed and evaluated for stem cell tracking. In this study, the size of the synthesized GdH-NPs was about 140 nm, and it had greater image enhancement ability than commercial gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). From the biocompability test, we found GdH-NPs were nontoxic for human mesenchymal stem cells (hMSCs). The expression of surface antigens of hMSCs after culture with GdH-NPs was examined, and it showed no difference from the control group. The results of transmission electron microscopy (TEM) imaging for labeled hMSCs showed GdH-NPs were accumulated in the cells by the endocytotic pathway. The accumulation of GdH-NPs in hMSCs was three times higher in comparison to Gd-DTPA. Human MSCs labeled with low concentration of GdH-NPs (10 mg/mL) hold better signals in cellular MR image. We conclude GdH-NPs can be used to label hMSCs in vitro with greater T1 image-enhancing property and without affecting cell quality. Finally, GdH-NPs have great potential as a contrast agent for stem cell tracking by MRI methodology. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Gadolinium Nanoparticle Stem cell Magnetic resonance imaging (MRI) Cell tracker
1. Introduction The ability to trace transplanted cells in vivo and monitor their tissue biodistribution is prerequisite to an understanding of cellular migration after transplantation [1,2], especially human stem cells, because of their multipotent ability to differentiate into various cell lineages for clinical applications [3,4]. Mesenchymal stem cells (MSCs) are an obvious source of autologous cells for organ repair or regeneration. However, the actual function and movement of stem cells after they are grafted or injected into the human body remains unknown. To determine the function and movement of therapeutic stem cells, it is crucial to develop a technique to trace them in vivo. Many techniques have been developed for tracking cells in vivo, such as fluorescence imaging, ultrasound, isotope labels, and magnetic resonance imaging (MRI) [5]. Fluorescence imaging has
* Corresponding author at: Division of Medical Engineering Research, National Health Research Institutes, No.35, Keyan Rd, Zhunan Town, Miaoli County 350, Taiwan, ROC. Tel.: þ886 37 246166x37100; fax: þ886 37 586440. E-mail address: [email protected] (F.-H. Lin). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.049
the limitation of detection depth for inner organs, and it also requires tissue samples for detection [6]. Limitations of echocardiography include accuracy in cell quantification and special resolution [5e7]. Nuclear medicine techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), require isotopes for labeling and could lead to radiation injury [8]. Limitations on the widespread use of PET and SPECT arise from the high costs of cyclotrons needed to produce the short-lived radionuclides required for scanning and the need for specially adapted on-site chemical synthesis apparatus to produce the radiopharmaceuticals, which may cause nuclear damage. Spatial resolution of these modalities is low and the definition of anatomy is poor. MRI has been widely studied as a cell tracking technique in recent years [9e11]. It is not only a noninvasive technique to monitor the movement of cells in vivo but also provides real-time images of the cells in internal organs. To obtain better MRI results, the selection of contrast agents is a key factor. The T2 contrast agents such as superparamagnetic iron oxide (SPIO) enhance an image with dark spots making it difficult to distinguish between cells in tissue with a low intrinsic MR signal and a hemorrhage condition. Conversely, contrast agents with
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Table 1 The concentrations of the antibodies used for immunophenotyping. Antibody
Fluorochrome
Dilution (mg/106 cells per ml)
CD29 CD44 CD45 CD71 CD105 CD106
FITC FITC FITC FITC PE PE
1.0 0.5 0.25 1.0 0.5 0.125
Table 2 Characterization of GdH-NPs. Brij 78 (mM)
4 6 10 30
GdH-NPs Size (nm)
Zeta (mV)
138.09 6.90 140.17 7.00 205.70 0.29 1131.22 56.56
3.8 1.1 0.4 1.2 1.6 0.8 1.9 0.3
FITC, fluorescein isothiocyanate; PE, phycoerythrin. Maker: eBioscience.
GdH-NPs: gadolinium hexanedione nanoparticles Brij 78: polyoxyethylene (20) stearyl ether.
T1-weighted enhancing ability produce bright positive signal intensity in images and increase the conspicuousness of cells, facilitating easy tracking of cells in low-signal tissues. Among those, gadolinium (Gd) is the most effective T1 contrast agent for clinical use. Gd (III) usually forms a complex with the chelating ligand, diethylenetriaminepentaacetic acid (DTPA) to give Gd-DTPA, which is used for MRI imaging. Although the hydrophilic property of GdDTPA leads to shortened clearance time in blood, it cannot pass through the cell membrane easily, and being an extracellular contrast agent, it has no cellular targeting ability [12]. Considerable efforts have been devoted to improving cell uptake and optimization of the relaxation effect using Gd-based nanoparticles (NPs) [1,13]. However, most studies with Gd-loaded NPs internalized in cells exhibited only moderate T1 enhancement [13,14]. Cellular tracking by these NPs often suffers from low cellular internalization, which manifests as the requirement for long-term incubation and high concentrations of NPs in cells for MR detection [15,16]. Because the intracellular concentration of Gd is critical to the success of MRI, engineered NPs containing higher concentrations of Gd in the form of gadolinium hexanedione (GdH) were sought [17]. For the purpose of stem cell tracking, in this study we have developed gadolinium hexanedione nanoparticles (GdH-NPs) with hydrophobic properties to enable it to easily merge with cell membranes, facilitating cell internalization. The GdH-NPs were
fabricated using the microemulsion process. The characteristics, biocompatibility, and T1-enhancing ability of these developed NPs were analyzed and evaluated as a suitable T1-MRI contrast agent for stem cell tracking. 2. Materials and methods 2.1. Reagent and chemicals GdCl3, 3, 4-hexanedione, polyoxyethylene (20) stearyl ether (Brij 78), alphaminimum essential medium (a-MEM), Ethylenediaminetetraacetic acid (EDTA), HistopaqueÒ-1077 were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Emulsifying wax was supplied by GBhouse (Taipei, Taiwan). Fetal bovine serum (FBS) was provided by Gemini Bio-Products (West Sacramento, CA USA), L-glutamine, trypsin-EDTA and antibiotic-antimycotic solution were obtained from Gibco/ BRL (Gaithersburg, MD, USA). Gd-DTPA (Omniscan) was purchased from AmershamNycomed (Oslo, Norway). WST-1, (4-[3-(4-iodophenyl)-2-(4-nitro phenyl)- 2H-5tetrazolio]-1, 3-benzene disulfonate), was obtained from Roche (Mannheim, Germany). CytoTox 96Ò assay kit was supplied by Promega (Madison, WI, USA). All other chemicals were from SigmaeAldrich in reagent grade. 2.2. Preparation of gadolinium hexanedione nanoparticles 2.2.1. Gadolinium hexanedione synthesis 5 g GdCl3 was dissolved in 40 mL deionized water, and then 6 mL of 3, 4-hexanedione solution (pH 2.5) was added into it. The mixture was stirred at 25 C to form a complex of Gd3þ and the dicarbonyl group of 3,4-hexanedione [17,18]. Thereafter, NaOH solution was used to adjust the pH value to 7.4, causing GdH
Fig. 1. Schematic depiction of the gadolinium hexanedione (GdH) nanoparticles preparation. (a) GdH synthesis and (b) oil-in-water microemulsion of GdH nanomicelle synthesis.
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Fig. 3. (a) MRI image of GdH-NPs and Gd-DTPA and (b) MRI signal intensity of GdHNPs and Gd-DTPA presented by the gray-level values. water was added to achieve a final volume of 25 mL and these oil-in-water microemulsion droplets were cooled to room temperature to obtain GdH-NPs [17]. 2.3. Characterization of nanoparticles
Fig. 2. (a) Transmission electron micrograph (TEM) and (b) scanning electron micrograph (SEM) showing the size and morphology of gadolinium hexanedione nanoparticles.
precipitation. The GdH was repeatedly washed with deionized water and purified by filtration to remove the excess agent. 2.2.2. Preparation of GdH nanoparticles GdH-NPs were obtained using the oil-in-water microemulsion technique. GdH and emulsifying wax were used as the oil phase, and polyoxyethylene (20) stearyl ether (Brij 78) was used as the surfactant in the oil-in-water microemulsion. Briefly, equal amounts of GdH and emulsifying wax were mixed and then melted at 60 C. The melted mixture was continuously stirred, and then the neutral surfactant (Brij 78) was added into the oil phase. Various concentrations of Brij 78 were tested in this experiment to evaluate the surfactant effect of the prepared NPs. Finally, warm Table 3 Element analysis of GdH nanoparticles. Element
Weight %
Atomic %
OK GdL AuM
35.65 49.86 14.49
85.08 12.11 2.81
2.3.1. Particle size, zeta potential, and morphology analyses The particle size of the GdH-NPs was determined by photon correlation spectroscopy (PCS) (Zetasizer-3000HS, Malvern, UK) at 25 C with scattering light at 90 and 180 s for each measurement. The NP suspension was diluted to ensure that the light scattering signal, as indicated by the particle counts per second, was within the sensitivity range. The zeta potential of the NPs was measured by the automatic mode to confirm the surface charge of the particles. The size and structure of the GdH-NPs were observed by a transmission electron microscope (TEM) (Hitachi, H-7500, Japan). One drop of GdH-NPs suspension was deposited on the copper spacemen grid. The grid was dried before examination. A field emission scanning electron microscope (FE-SEM) (Hitachi, S4100, Japan), equipped with an energy dispersive spectrometry (EDS) microprobe analyzer was adopted to quantify the Gd amount in the GdH-NPs. The colloidal solution was loaded on the cover slide and dried; then, the surface of the specimenwas coated with a thin gold film by sputtering physical vapor deposition (PVD). 2.3.2. MRI of GdH-NPs The GdH-NPs and Gd-DTPA were diluted to Gd concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg/mL with deionized water and transferred to a microtiter tube. MRI examination of these solutions was performed by MR imagery (Magnetom, Trio 3-Tesla (3 T) MRI, Siemens, Germany) with a T1-weighted spin-echo sequence (repetition time (TR) ¼ 150 ms, (echo time) TE ¼ 7.8 ms, voxel size ¼ 0.9 0.9 1, slice ¼ 15, number of excitations (NEX) ¼ 2, resolution ¼ 256). 2.4. In vitro cell culture test of nanoparticles 2.4.1. Human mesenchymal stem cell culture Human mesenchymal stem cells (hMSCs) were isolated from the bone marrow of healthy donors after informed consent. The bone marrow mesenchymal stem
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C.-L. Tseng et al. / Biomaterials 31 (2010) 5427e5435 Triton-X 100 (total lysis group) was adopted as the positive control. After 1, 3, and 7 days of incubation, the culture medium was assayed by the LDH assay kit. In brief, 50 ml of the medium was transferred to a new enzymatic assay plate in which 50 ml of the LDH substrate solution was added. After 30 min, 50 ml of stop solution was added to each well in the plate and read on microplate reader spectrophotometer (SpectraMAX M2; Molecular Devices, USA) at 490 nm optical density (OD). All experiments were repeated 6 times for statistical analysis. 2.5. Nanoparticle cell uptaken study 2.5.1. TEM analysis of cells with internalized nanoparticles TEM was used for the observation of intracellular NPs distribution. Human MSCs were exposed to GdH-NPs suspension for 3 h at 37 C at a concentration of 100 mg/ mL. These hMSCs were washed with PBS 3 times, and then fixed at 4 C in a solution of 2.5% buffered glutaraldehyde for 8 h, then post-fixed in 1% osmium tetraoxide for 2 h, and then washed and dehydrated with increasing concentrations of ethanol and propylene oxide. Cells were embedded in Spurr’s resin and cut into ultrathin sections (50w100 nm). These sections were collected on copper grids and stained with a 1:1 mixture of methanol and lead citrate. The grids were examined using an H-7500 TEM (Hitachi, Japan). 2.5.2. Intracellular Gd accumulation To quantify intracellular Gd concentration, an inductively coupled plasma-mass spectrometer (ICP-MS) (Agilent 7500C, USA) was used in this study. Human MSCs were grown in 10-cm culture dishes. These cells were incubated with GdH-NPs and Gd-DTPA for 12 h at 37 C with a Gd concentration of 20 mg/mL. In control cultures, the cells were placed in a medium without GdH-NPs at the same cell density. Cells were washed 3 times with PBS and then detached by trypsinization. The labeled cell pellets were treated with 65% HNO3 solution at 80 C for 60 min. The samples were diluted to the proper range for analysis. 2.5.3. In vitro MRI of Gd-labeled stem cells To evaluate the GdH-NPs contrast in T1-weighted sequences, cells were incubated with GdH-NPs at concentrations of 10 and 100 mg/mL GdH-NPs for 24 h. Subsequently, cells were trypsinized and washed twice with the culture medium by centrifugation at 1500 rpm for 5 min. After each step the supernatant was harvested again for subsequent MRI examinations to ensure that no GdH-NPs were left in the medium after washing. After the final centrifugation, the cell pellet was analyzed using MRI. MRI was performed using a Magnetom Trio 3-T MRI system with a T1weighted spin-echo sequence (repetition time (TR) ¼ 150 ms, (echo time) TE ¼ 7.8 ms, voxel size ¼ 0.9 0.9 1, slice ¼ 15, number of excitations (NEX) ¼ 2, resolution ¼ 256). To avoid susceptibility artifacts from the surrounding air in the scans, all samples were placed in a water-containing plastic container and evaluated at room temperature. 2.6. Characterization of hMSCs
Fig. 4. The biocompatibility of GdH-NPs for MSCs cultivation examined by the (a) WST-1 and (b) LDH assay with different Gd concentrations after 1, 3, and 7 days of treatment (one-way ANOVA, mean SD, n ¼ 6, *p < 0.05: significantly different). cells (MSCs) were isolated by density gradient centrifugation (1.077 g/cm3, Histopaque) and washed in phosphate buffer saline (PBS). Human MSCs were cultured in alpha-minimum essential medium (a-MEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% antibiotic-antimycotic solution, which was the complete a-MEM medium. When the cultures reached 85% confluence, the cells were detached with trypsin-EDTA solution and subcultured at 1 104 cells/mL. 2.4.2. Cell viability Cell proliferation reagent WST-1 kit, was used to examine cell viability. The cells were seeded in 96-well plates at a density of 5 103 cells/well. Cells were incubated with different concentrations of GdH-NPs suspension. The concentrations of GdHNPs were tested at 0, 30, 50,100, and 300 mg/mL. After culturing hMSCs with GdH-NPs for 1, 3, and 7 days, WST-1 reagent was added for cell proliferation assay. After incubation with WST-1 for 2 h at 37 C, the formazan dye generated by the activity of dehydrogenases in cells was proportional to the number of living cells; the absorbance at 420 nm was measured using a microplate reader (SpectraMAX M2; Molecular Devices, USA). All experiments were repeated 6 times for statistical analysis. 2.4.3. Cell toxicity Lysed cells could release lactate dehydrogenase (LDH). Therefore, cell toxicity was evaluated by extracellular LDH content. The LDH was measured by a commercial assay kit (CytoTox 96Ò Assay, Promega, USA). The hMSCs were seeded in 96-well plates at a density of 5 103 cells/well. After a 24-h incubation, the cells were incubated with different concentrations of GdH-NPs (30, 50, 100, and 300 mg/mL). Cells incubated in a medium without GdH-NPs were used as the control group, and
Human MSCs were seeded at a density of 1 106 cells per culture dish (10-cm dishes) and allowed to attach for 24 h. After that, the cells were incubated with GdHNPs (100,10 mg/mL) for an additional 24 h. At the end of incubation, cells were washed 3 times with PBS to remove free GdH-NPs and were harvested using trypsinEDTA. The harvested cells were collected and stained either with fluorescein isothiocyanate (FITC)-conjugated antibodies against human antigens CD29, CD44, CD45,and CD71, or with phycoerythrin (PE)-conjugated antibodies against human antigens CD105 and CD106 (eBioscience Inc, San Diego, CA, USA) [16,19] as described in Table 1. Unlabeled cells are used as a control and isotype antibodies are used, respectively, to exclude nonspecific staining. The cells are incubated at 4 C to diminish the possible nonspecific staining caused by temperature. The incubation should be maintained in the dark to avoid fluorescent attenuation. The cells were examined using flow cytometry (FACSCalibur, Becton Dickinson, USA) to confirm the stem cells’ surface marker. Positive staining was defined as the fluorescence emission that exceeded levels obtained by more than 99% of the cells from the population stained with isotype-matched controls.
3. Results 3.1. Size and morphology of GdH-NPs In this study, the oil-in-water microemulsion system was used to prepare GdH-NPs (Fig. 1). Table 2 shows the size distribution of GdH-NPs prepared in different surfactant (Brij 78) concentrations. Particles size of GdH-NPs prepared by using 4 mM Brij 78 was 138.09 6.90 nm. The particle size at the concentrations of 6, 10, and 30 mM of Brij 78 were 140.17 7.00, 205.70 0.29, and 1131.22 56.56 nm, respectively. There was an enormous increase in the mean particle size from 138.09 nm to 1131.22 nm. The GdH-
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Fig. 5. Surface marker identification of hMSCs (a) before and (b) after GdH-NP cultivation for 24 h at a concentration of 100 mg/mL.
NPs made using 30 mM Brij 78 had 2 major size distributions in the raw data. One was 245.2 45.8 nm and the other was 1459.1 406.4 nm. Because the concentrations of the surfactants are key parameters in preparing NPs by the microemulsion technique, the surface charge of these particles was affected by surfactant concentration. The GdH-NPs made using 4 mM Brij 78 led to the highest zeta potential, 3.8 1.1 mV (Table 2). Other
concentrations of the surfactant affected the zeta potential, producing values that range from 1.6 to þ3.8 mV. Nanoparticles are more stable when the zeta potential is higher. Smaller particle size and higher zeta potential values were observed in the group of the 4 mM Brij 78. The GdH-NPs prepared using the oil-in-water microemulsion template at the concentration of 4 mM Brij 78 was used in the following experiment.
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Fig. 7. Intracellular Gd concentration examined by ICP-MS assay.
3.2. GdH-NPs as an MRI contrast agent Fig. 3(a) shows T1-weighted spin-echo MR images of two kinds of contrast agents, the GdH-NPs, made from microemulsion method, and Gd-DTPA. Both solutions were diluted into different concentrations ranging from 200 to 6.25 mg/mL. The MRI image shows that GdH-NPs produced stronger signal intensity than GdDTPA. The gray-level values of the MRI images are presented in Fig. 3(b). It shows significant differences in gray-level values resulting from GdH-NPs and Gd-DTPA at 50, 100, and 200 mg/mL concentrations. The gray-level value of GdH-NPs at 200 mg/ml was 2.2 times higher than that of Gd-DTPA. 3.3. The biocompatibility of GdH-NPs
Fig. 6. TEM images show the GdH nanoparticles (100 mg/mL, 37 C, and 3 h) internalized into the hMSC cells. (a) Pseudopods were observed at the cell surface (indicated by arrows) and (b) GdH were found in endosomes (indicated by arrows).
Two kinds of electron microscope were used to examine the size and structure of GdH-NPs. Fig. 2(a) was acquired by TEM and Fig. 2 (b) was obtained by SEM. Both figures show that the GdH-NPs were well dispersed and no aggregation was observed. The particle size was about 100 nm. These results are in agreement with the particle size analysis (Table 2). In Fig. 2(a), it can be seen that the central part of the NPs was dark, which may have been caused by Gd encapsulation. SEM equipped with an EDS microprobe analyzer was used to quantify the Gd amount in the NPs. Table 3 provides the results of element analysis corresponding to Fig. 2(b), which illustrates GdH-NPs composted with element of Gd with it. These results indicate that the microemulsion method can be used to prepare nano-scale particles.
To evaluate possible cytotoxic effect of GdH-NPs, cell viability was examined by WST-1 reduction assay. No evidence of cytotoxicity was observed after 1, 3, and 7 days of cultivation with GdH-NPs at concentrations ranging from 30 to 300 mg/mL ((Fig. 4(a)). The viability of hMSCs treated by GdH-NPs at different concentrations was not significantly different from that of the control group (oneway ANOVA, p < 0.05). These data indicated that GdH-NPs were not toxic and were biocompatible for hMSC culture at the tested concentrations. LDH is a stable cytosolic enzyme that is released into the culture supernatant during cell lysis. This may be used to determine the cytotoxicity of cells influenced by different additives or culture conditions. In our study, after incubation with GdH-NPs for 1, 3, and 7 days, the LDH released by cultured hMSCs was collected and measured. The OD value in spontaneous release of LDH from hMSCs with NP treatment was regarded as the control and medium containing Triton X-100 was considered as the positive control (total lysis group). Fig. 4(b) shows that GdH-NPs mediated very low cytotoxicity under concentrations ranging from 30 to 300 mg/mL after co-culture with hMSCs at the indicated times. These results are in agreement with those of the WST-1 test. There was no significant difference between the control and GdH-NPs concentrations ranging from 30 to 300 mg/mL. 3.4. MSC characterization after GdH-NPs co-culture To exclude possible differentiation effects of GdH-NPs on the function of stem cells, we examined the immunophenotypes of labeled hMSCs. As shown in Fig. 5, cell surface antigen profiles of hMSCs were positive for primitive mesenchymal progenitor cell markers, including CD29, CD44, CD71, CD105, and CD 106, whereas
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strong signal intensity change but not the typical bright signal. The MRI image acquired by GdH-NPs (100 mg/mL)-treated group showed a dark image (Fig. 8(b)). 4. Discussion
Fig. 8. Representative T1-weighted MR images of Eppendorf test tubes that contained centrifuged cell pellets after labeling with GdH-NPs in different concentrations for 24 h. (a) hMSCs without labeling, (b) 100 mg/mL and (c) 10 mg/mL GdH-NP labeling.
the hematopoietic lineage-specific marker CD45 was not detected. Other surface markers of hMSCs labeled with GdH-NPs were not affected. Although CD71 and CD106 expression slightly increased after GdH-NPs treatment (11.0 7.94% to 25.5 8.97%; 16.9 12.93% to 30.8 11.51%), no significant difference was observed (One-way ANOVA, mean SD, p < 0.05). 3.5. Intracellular distribution and accumulation of GdH-NPs TEM was carried out to confirm bioadhesion and endocytosis of GdH-NPs with cells. Micrographs of hMSCs incubated with GdHNPs at Gd concentration of 100 mg/mL for 3 h at 37 C are shown in Fig. 6. These micrographs clearly indicated that GdH-NP adhered to hMSCs and the membrane invaginated, forming a pocket containing NPs (Fig. 6(a)). Some particles were incorporated within the cells, and endocytic vesicles (indicated by arrows) with GdH-NPs in the cytosol were also found (Fig. 6(b)). 3.6. Quantification of intracellular Gd concentration Fig. 7 provides the quantitative analysis of the amount of Gd in cells as a contrast agent, with GdH uptake by hMSCs after 24-h cultivation. The results of cells treated with GdH-NPs shows that the Gd concentration in hMSCs was 3.25-fold higher than that in the Gd-DTPA-treated group (3.209 mg/mL versus 1.034 mg/mL, respectively). A one-way ANOVA test showed a significant difference between Gd-DTPA- and GdH-treated groups. The intracellular Gd concentration of hMSCs treated with GdH-NPs was significantly higher than those treated with Gd-DTPA (one-way ANOVA, p < 0.05). 3.7. Cellular MRI imagery We analyzed what concentration of GdH-NPs provided the strongest contrast of hMSCs cells in vitro (Fig. 8(aec)).We found that coincubation of GdH-NPs with hMSCs at 10 mg/mL for 24 h provided the strongest signal of the cell pellet in T1-weighted MRI with bright image (Fig. 8(c))(compared with nonlabeled one, Fig. 8 (a)). Human MSCs cells labeled with 100 mg/mL GdH-NPs showed
For clinical research, transplantation of stem cells for cell therapy needs to be monitored. Labeling of stem cells has become a frequently applied technique to investigate the aforementioned biological properties of stem cells. This study showed that hMSCs incubated with GdH-NPs spontaneously internalized this hydrophobic, nano-sized contrast agent. Cellular uptake after incubation with GdH-NPs was sufficient for the tracking of hMSCs by using T1weighted sequences with clinical MR scanners without toxicity, and would not induce differentiation of hMSCs. MRI has been widely studied as a cell tracking technique in recent years. It has good spatial resolution but in order to become suitable as a cellular imaging modality, its inherently low sensitivity must be dealt with. This may be overcome by using contrast agents with very high relaxivity matter (e.g., by using ferric oxide NPs or a high payload of Gd complexes). In biological tissues, T1 relaxivity is longer than that of T2; therefore, contrast enhancement is better observed with T1-weighted sequences [20]. Moreover, T1-sensitive contrast agents can monitor the distribution of freshly implanted cells more accurately than T2-wighted ferric oxide NP-labeled contrast agents [21]. Gd is the most effective T1 contrast agent for clinical use. Therefore, incorporating Gd3þ into cells for transplantation will change their relaxation characteristics, making it possible to distinguish between labeled and non-labeled cells [22]. Previous studies have shown that lanthanides complexes with dicarbonyl chelating agents have favorable physical properties, thermal stability, and consist of a high weight percentage of the lanthanide metal [23]. In this study, GdCl3, a lanthanides complex, reacted with 3, 4 hexandione (with dicarbonyl compound) to form GdH with hydrophobicity [17]; this is a stable Gd complex that could reduce the free toxic Gd released by unstable Gd-chelate in vivo. Compared to other complexes of Gd (such as Gd-DTPA, a hydrophilic form), GdH with high hydrophobicity could facilitate it with high affinity to phospholipid cell membrane, thus increasing the Gd content in cells [21]. Therefore, GdH-NP was the final NP formulation choice in this study. Methods to enhance MR imaging quality by increasing the imaging ability of contrast agents are as follows: (a) increase the water content in the Gd-complex to increase the water exchanging rate, (b) increase the molecular weight of the Gd-complex to change the Gd- complex relaxivity for increasing the relaxivity rate, and (c) decrease the tumbling rate of the Gd-complex [20]. In this study, the GdH-NPs fabricated by oil-in-water microemulsion method could encapsulate more Gd-chelate in it to form larger Gdcomplexes with high molecular weight causing the slow tumbling rate of GdH-NPs, thus being responsible for the increase in T1 relaxivity that was observed in the strong intensity of the MRI image [2,24]. Therefore, it had better images quality. For stem cell tracking, the safety effect of internalized GdH-NPs should first be investigated. Free Gd ion is more toxic to the human body [2]; therefore, applied Gd-based contrast agents are chelated to form Gd-complex, which increases the biological safety. However, intracellular dechelation of the Gd-DTPA complex liberates the highly toxic Gd ion and causes toxicity. To evaluate possible cytotoxicity effects of GdH-NPs, cell viability and toxicity were examined by WST-1 and LDH assay. Results obtained from these assays (Fig. (4)(a) and (b)) show that the GdH-NPs at the tested concentrations (30 to 300 mg/ml) produced no toxicity. Owing to the result that GdH-NPs at the concentration of 200 mg/ml had good
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T1-weighted signal enhancement from MRI examination (Fig. 3), it could easily be adapted for stem cell labeling. One unexpected outcome of stem cell culture is that typical hMSCs markers such as CD44, CD90, and CD105 can be irrecoverably lost during improper cultivation. After GdH-NPs treatment, the immunotype of hMSCs such as CD29, CD44, CD71, CD105, and CD106 were not affected (Fig. 5) demonstrating hMSCs were not differentiation. Efficient magnetic labeling of cells to be tracked with MRI is of great importance for keeping the detection limit of the cells as low as possible. The uptake of contrast agents by cells can occur via the endocytosis [25]. For endocytosis, the invagination of particles on the cell membrane could forms a vesicle. The hydrophobic property and nano-size of GdH-NPs would facilitate cellular uptake because of the better affinity between GdH-NPs and cell membranes, which enhances transmembrane transport. TEM was utilized to confirm the endocytosis of GdH-NPs (Fig. 6(a), (b)). GdH-NPs were engulfed by cell membrane and entrapped in the endosome. The intracellular Gd accumulation also confirmed that hydrophobic GdH-NPs more easily came into contact with cell membrane and were uptaken into cytoplasm, causing higher Gd accumulation in cells than did Gd-DTPA (Fig. 7). These results agree with a previous study that the amphiphilic structure allows the gadofluorine M molecule to penetrate phospholipid bilayers and to interact with intracellular water protons, which increases R1 relaxation times and leads to the observed increased signal on T1-weighted images [21]. At low concentration of GdH-NPs there will be an increase in signal intensity via T1-weighted signal. From Fig. 3, it shows that GdH-NPs have a superior T1-enhancing ability as compared to that of GdDTPA, and it could improve cellular MRI by using lower Gd concentrations (Fig. 8). Otherwise in Fig. 8(b), hMSCs labeled with 100-mg/ml GdH-NPs caused the T2-weighted image, the dark enhancement. This may have been owing to the high concentration of GdH-NPs labeled on hMSCs; when the concentration of Gd is too high, the effect of T2 relaxation will overcome the effect of T1 and this will suppress the signal of T1 [26]. In this study, we successfully labeled hMSCs with GdH-NPs at a lower Gd concentration but with better T1-weighted MRI images (Fig. 8(c)). Cells that lack substantial phagocytic capacity, such as stem cells, are more difficult to label [21]. One was to use anionic hydrophobic NPs that interact strongly and nonspecifically with the cell plasma membrane because of strong negative surface charges, and easily combine with the lipid cell membrane [27]. The use of hydrophobic molecules, like GdH, might provide a considerable advantage because these molecules can be expected to work with most cell types for endocytosis. Another method to improve hMSCs T1-weighted signal cellular uptake was to couple NPs to antibodies, which penetrate the cell membrane by receptor-mediated internalization [21,28]. Labeling of stem cells via receptor-mediated internalization of molecules might affect the physiological properties of stem cells, such as proliferation, differentiation, or cellecell interactions resulting from the blockade of important receptor-epitopes. Furthermore, it might be problematic if the antibodies will not stick to the NPs surface for a prolonged period of time. 5. Conclusion In this study we successfully synthesized GdH-NPs with promising T1-weighted enhancing properties for MRI applications. The particle size was about 140 nm based on PCS and TEM examination. MRI showed that GdH-NPs have a much higher gray-level signal as compared to Gd-DTPA. From the in vitro cell culture tests, we found that GdH-NPs were biocompatible and were taken up by endocytosis. The intracellular Gd accumulation was much higher in GdHNPs-treated cells than in Gd-DTPA-treated cells. Furthermore,
labeled stem cells showed better signals in cellular MRI assay. The expression of surface antigens of hMSCs after culture with GdH-NPs was examined, and it showed no difference from the control group. We believe that, in the near future, the GdH-NPs developed in this study have great potential as a contrast agent for stem cell tracking by MRI methodology. Appendix Figures with essential color discrimination. Figs. 1 and 5 in this article are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.biomaterials.2010.03.049. References [1] Vuu K, Xie J, McDonald MA, Bernardo M, Hunter F, Zhang Y, et al. Gadoliniumrhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjugate Chem 2005;16:995e9. [2] Mulder WJ, Strijkers GJ, vanTilborg GA, Griffioen AW, Nicolay K. Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed 2006;19:142e64. [3] Pittenger M, Mackay A, Beck S, Jaiswal R, Douglas R, Mosca J, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284 (5411):143e7. [4] Barry F, Murphy J. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36(4):568e84. [5] Chemaly ER, Yoneyama R, Frangioni JV, Hajjar RJ. Tracking stem cells in the cardiovascular system. Trends Cardiovasc Med 2005;15(8):297e302. [6] Hardy J, Edinger M, Bachmann M, Negrin R, Fathman C, Contag C. Bioluminescence imaging of lymphocyte trafficking in vivo. Exp Hematol 2001;29 (12):1353e60. [7] Morawski AM, Lanza GA, Wickine SA. Target contrast agents for magnetic resonance imaging and ultrasound. Curr Opin Biotechnol 2005;16:89e92. [8] Adonai N, Nguyen K, Walsh J, Iyer M, Toyokuni T, Phelps M, et al. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A 2002;99(5):3030e5. [9] Hinds K, Hill J, Shapiro E, Laukkanen M, Silva A, Combs C, et al. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 2003;102(3):867e72. [10] Lanza GM, Winter PM, Caruthers SD, Morawski MA, Schmieder AH, Crowder KC, et al. Magnetic resonance molecular imaging with nanoparticles. J Nucl Cardiol 2004;11(6):733e43. [11] Leslie LC, Nitin N, Gang B. Magnetic nanoparticle probes. Mat Today 2005;8 (5):32e8. [12] Hengerer A, Grimm J. Molecular magnetic resonance imaging. Biomed Imaging Interv J 2006;2(2):e8. [13] Hsiao J-K, Tsai C-P, Chung T-H, Hung Y, Yao M, Liu H-M, et al. Mesoporous silica nanoparticles as a delivery system of gadolinium for effective human stem cell tracking. Small 2008;4:1445e52. [14] Prantner AM, Sharma V, Garbow JR, Piwnica-Worms D. Synthesis and characterization of a Gd-DOTA-D-permeation peptide for magnetic resonance relaxation enhancement of intracellular targets. Mol Imaging 2003;2:333e41. [15] Hill JM, Dick AJ, Raman VK, Thompson RB, Yu ZX, Hinds KA. Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009e14. [16] Croft AP, Przyborski SA. Mesenchymal stem cells from the bone marrow stroma: basic biology and potential for cell therapy. Curr Anaesth Crit Care 2004;15:410e7. [17] Oyewumi M, Mumper R. Engineering tumor-targeted gadolinium hexanedione nanoparticles for potential application in neutron capture therapy. Bioconjug Chem 2002;13(6):1328e35. [18] Mumper RJ, Mills BJA, Ryo UY, Jay M. Polymeric microspheres for radionucide synovectomy containing neutron-activated Holmium-166. J NucI Med 1992;33:398e402. [19] Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: Paradoxes of passaging. Exp Hematol 2004;32:414e25. [20] Tilcock C, Unger E, Cullis P, MacDougall P. Liposomal Gd-DTPA: Preparation and charaterization of relaxivity. Radiology 1989;171:77e80. [21] Nolte IS, Gungor S, Erber R, Plaxina E, Scharf J, Misselwitz B, et al. In vitro labeling of glioma cells with gadofluorine M enhances T1 visibility without affecting glioma cell growth or motility. Magn Res Med 2008;59:1014e20. [22] Modo M, Cash D, Mellodew K, Williams SCR, Fraser SE, Meade TJ, et al. Tracking transplanted stem cell migration using bifunctional, contrast agentenhanced, magnetic resonance imaging. Neuro Image 2002;17:803e11. [23] Mumper RJ, Jay M. The formation and stability of lanthanide complexes and their encapsulation into polymeric microspheres. J Phys Chem 1992;96: 8626e31. [24] Artemov D, Bhujwalla Z, Bulte J. Magnetic resonance imaging of cell surface receptors using targeted contrast agents. Curr Pharm Biotechnol 2004;5(6):485e94.
C.-L. Tseng et al. / Biomaterials 31 (2010) 5427e5435 [25] Henning TD, Saborowski O, Golovko D, Boddington S, Bauer JS, Fu Y, et al. Cell labeling with the positive MR contrast agent Gadofluorine M. Eur Radiol 2007;17:1226e34. [26] Brasch RC. New directions in the development of MR imaging contrast media. Radiology 1992;183:1e11.
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[27] Riviere C, Boudghene F, Gazeau F, Roger J, Pons J, Laissy J, et al. Iron oxide nanoparticle-labeled rat smooth muscle cells: cardiac MR imaging for cell graft monitoring and quantitation. Radiology 2005;235:959e67. [28] Weissleder R, Moore A, Mahmood U, Bhorade R, Benveniste H, Chiocca E, et al. In vivo magnetic resonance imaging of transgene expression. Nat Med 2000;6: 351e5.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Gadolinium hexanedione nanoparticles for stem cell labeling and tracking via magnetic resonance imaging Ching-Li Tseng a, I.-Ling Shih b, Leszek Stobinski c, Feng-Huei Lin a, b, * a
Division of Medical Engineering Research, National Health Research Institutes, No.35, Keyan Rd, Zhunan Town, Miaoli County 350, Taiwan, ROC Institute of Biomedical Engineering, National Taiwan University, No.1, Sec. 1, Ren-ai Rd., Taipei City 100, Taiwan, ROC c Collage of Mechanical and Electrical Engineering, National Taipei University of Technology, Taipei, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2010 Accepted 19 March 2010 Available online 18 April 2010
The ability to trace transplanted stem cells and monitor their tissue biodistribution is prerequisite to an understanding of cellular migration after transplantation. Therefore, a new magnetic resonance imaging (MRI) contrast agent made of gadolinium hexanedione nanoparticles (GdH-NPs) was developed as a cell tracking agent. The GdH-NPs were fabricated by the microemulsion process. The physical characteristics, biocompatibility, and T1-MRI signal enhancement of these NPs were analyzed and evaluated for stem cell tracking. In this study, the size of the synthesized GdH-NPs was about 140 nm, and it had greater image enhancement ability than commercial gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). From the biocompability test, we found GdH-NPs were nontoxic for human mesenchymal stem cells (hMSCs). The expression of surface antigens of hMSCs after culture with GdH-NPs was examined, and it showed no difference from the control group. The results of transmission electron microscopy (TEM) imaging for labeled hMSCs showed GdH-NPs were accumulated in the cells by the endocytotic pathway. The accumulation of GdH-NPs in hMSCs was three times higher in comparison to Gd-DTPA. Human MSCs labeled with low concentration of GdH-NPs (10 mg/mL) hold better signals in cellular MR image. We conclude GdH-NPs can be used to label hMSCs in vitro with greater T1 image-enhancing property and without affecting cell quality. Finally, GdH-NPs have great potential as a contrast agent for stem cell tracking by MRI methodology. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Gadolinium Nanoparticle Stem cell Magnetic resonance imaging (MRI) Cell tracker
1. Introduction The ability to trace transplanted cells in vivo and monitor their tissue biodistribution is prerequisite to an understanding of cellular migration after transplantation [1,2], especially human stem cells, because of their multipotent ability to differentiate into various cell lineages for clinical applications [3,4]. Mesenchymal stem cells (MSCs) are an obvious source of autologous cells for organ repair or regeneration. However, the actual function and movement of stem cells after they are grafted or injected into the human body remains unknown. To determine the function and movement of therapeutic stem cells, it is crucial to develop a technique to trace them in vivo. Many techniques have been developed for tracking cells in vivo, such as fluorescence imaging, ultrasound, isotope labels, and magnetic resonance imaging (MRI) [5]. Fluorescence imaging has
* Corresponding author at: Division of Medical Engineering Research, National Health Research Institutes, No.35, Keyan Rd, Zhunan Town, Miaoli County 350, Taiwan, ROC. Tel.: þ886 37 246166x37100; fax: þ886 37 586440. E-mail address: [email protected] (F.-H. Lin). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.049
the limitation of detection depth for inner organs, and it also requires tissue samples for detection [6]. Limitations of echocardiography include accuracy in cell quantification and special resolution [5e7]. Nuclear medicine techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), require isotopes for labeling and could lead to radiation injury [8]. Limitations on the widespread use of PET and SPECT arise from the high costs of cyclotrons needed to produce the short-lived radionuclides required for scanning and the need for specially adapted on-site chemical synthesis apparatus to produce the radiopharmaceuticals, which may cause nuclear damage. Spatial resolution of these modalities is low and the definition of anatomy is poor. MRI has been widely studied as a cell tracking technique in recent years [9e11]. It is not only a noninvasive technique to monitor the movement of cells in vivo but also provides real-time images of the cells in internal organs. To obtain better MRI results, the selection of contrast agents is a key factor. The T2 contrast agents such as superparamagnetic iron oxide (SPIO) enhance an image with dark spots making it difficult to distinguish between cells in tissue with a low intrinsic MR signal and a hemorrhage condition. Conversely, contrast agents with
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Table 1 The concentrations of the antibodies used for immunophenotyping. Antibody
Fluorochrome
Dilution (mg/106 cells per ml)
CD29 CD44 CD45 CD71 CD105 CD106
FITC FITC FITC FITC PE PE
1.0 0.5 0.25 1.0 0.5 0.125
Table 2 Characterization of GdH-NPs. Brij 78 (mM)
4 6 10 30
GdH-NPs Size (nm)
Zeta (mV)
138.09 6.90 140.17 7.00 205.70 0.29 1131.22 56.56
3.8 1.1 0.4 1.2 1.6 0.8 1.9 0.3
FITC, fluorescein isothiocyanate; PE, phycoerythrin. Maker: eBioscience.
GdH-NPs: gadolinium hexanedione nanoparticles Brij 78: polyoxyethylene (20) stearyl ether.
T1-weighted enhancing ability produce bright positive signal intensity in images and increase the conspicuousness of cells, facilitating easy tracking of cells in low-signal tissues. Among those, gadolinium (Gd) is the most effective T1 contrast agent for clinical use. Gd (III) usually forms a complex with the chelating ligand, diethylenetriaminepentaacetic acid (DTPA) to give Gd-DTPA, which is used for MRI imaging. Although the hydrophilic property of GdDTPA leads to shortened clearance time in blood, it cannot pass through the cell membrane easily, and being an extracellular contrast agent, it has no cellular targeting ability [12]. Considerable efforts have been devoted to improving cell uptake and optimization of the relaxation effect using Gd-based nanoparticles (NPs) [1,13]. However, most studies with Gd-loaded NPs internalized in cells exhibited only moderate T1 enhancement [13,14]. Cellular tracking by these NPs often suffers from low cellular internalization, which manifests as the requirement for long-term incubation and high concentrations of NPs in cells for MR detection [15,16]. Because the intracellular concentration of Gd is critical to the success of MRI, engineered NPs containing higher concentrations of Gd in the form of gadolinium hexanedione (GdH) were sought [17]. For the purpose of stem cell tracking, in this study we have developed gadolinium hexanedione nanoparticles (GdH-NPs) with hydrophobic properties to enable it to easily merge with cell membranes, facilitating cell internalization. The GdH-NPs were
fabricated using the microemulsion process. The characteristics, biocompatibility, and T1-enhancing ability of these developed NPs were analyzed and evaluated as a suitable T1-MRI contrast agent for stem cell tracking. 2. Materials and methods 2.1. Reagent and chemicals GdCl3, 3, 4-hexanedione, polyoxyethylene (20) stearyl ether (Brij 78), alphaminimum essential medium (a-MEM), Ethylenediaminetetraacetic acid (EDTA), HistopaqueÒ-1077 were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Emulsifying wax was supplied by GBhouse (Taipei, Taiwan). Fetal bovine serum (FBS) was provided by Gemini Bio-Products (West Sacramento, CA USA), L-glutamine, trypsin-EDTA and antibiotic-antimycotic solution were obtained from Gibco/ BRL (Gaithersburg, MD, USA). Gd-DTPA (Omniscan) was purchased from AmershamNycomed (Oslo, Norway). WST-1, (4-[3-(4-iodophenyl)-2-(4-nitro phenyl)- 2H-5tetrazolio]-1, 3-benzene disulfonate), was obtained from Roche (Mannheim, Germany). CytoTox 96Ò assay kit was supplied by Promega (Madison, WI, USA). All other chemicals were from SigmaeAldrich in reagent grade. 2.2. Preparation of gadolinium hexanedione nanoparticles 2.2.1. Gadolinium hexanedione synthesis 5 g GdCl3 was dissolved in 40 mL deionized water, and then 6 mL of 3, 4-hexanedione solution (pH 2.5) was added into it. The mixture was stirred at 25 C to form a complex of Gd3þ and the dicarbonyl group of 3,4-hexanedione [17,18]. Thereafter, NaOH solution was used to adjust the pH value to 7.4, causing GdH
Fig. 1. Schematic depiction of the gadolinium hexanedione (GdH) nanoparticles preparation. (a) GdH synthesis and (b) oil-in-water microemulsion of GdH nanomicelle synthesis.
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Fig. 3. (a) MRI image of GdH-NPs and Gd-DTPA and (b) MRI signal intensity of GdHNPs and Gd-DTPA presented by the gray-level values. water was added to achieve a final volume of 25 mL and these oil-in-water microemulsion droplets were cooled to room temperature to obtain GdH-NPs [17]. 2.3. Characterization of nanoparticles
Fig. 2. (a) Transmission electron micrograph (TEM) and (b) scanning electron micrograph (SEM) showing the size and morphology of gadolinium hexanedione nanoparticles.
precipitation. The GdH was repeatedly washed with deionized water and purified by filtration to remove the excess agent. 2.2.2. Preparation of GdH nanoparticles GdH-NPs were obtained using the oil-in-water microemulsion technique. GdH and emulsifying wax were used as the oil phase, and polyoxyethylene (20) stearyl ether (Brij 78) was used as the surfactant in the oil-in-water microemulsion. Briefly, equal amounts of GdH and emulsifying wax were mixed and then melted at 60 C. The melted mixture was continuously stirred, and then the neutral surfactant (Brij 78) was added into the oil phase. Various concentrations of Brij 78 were tested in this experiment to evaluate the surfactant effect of the prepared NPs. Finally, warm Table 3 Element analysis of GdH nanoparticles. Element
Weight %
Atomic %
OK GdL AuM
35.65 49.86 14.49
85.08 12.11 2.81
2.3.1. Particle size, zeta potential, and morphology analyses The particle size of the GdH-NPs was determined by photon correlation spectroscopy (PCS) (Zetasizer-3000HS, Malvern, UK) at 25 C with scattering light at 90 and 180 s for each measurement. The NP suspension was diluted to ensure that the light scattering signal, as indicated by the particle counts per second, was within the sensitivity range. The zeta potential of the NPs was measured by the automatic mode to confirm the surface charge of the particles. The size and structure of the GdH-NPs were observed by a transmission electron microscope (TEM) (Hitachi, H-7500, Japan). One drop of GdH-NPs suspension was deposited on the copper spacemen grid. The grid was dried before examination. A field emission scanning electron microscope (FE-SEM) (Hitachi, S4100, Japan), equipped with an energy dispersive spectrometry (EDS) microprobe analyzer was adopted to quantify the Gd amount in the GdH-NPs. The colloidal solution was loaded on the cover slide and dried; then, the surface of the specimenwas coated with a thin gold film by sputtering physical vapor deposition (PVD). 2.3.2. MRI of GdH-NPs The GdH-NPs and Gd-DTPA were diluted to Gd concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg/mL with deionized water and transferred to a microtiter tube. MRI examination of these solutions was performed by MR imagery (Magnetom, Trio 3-Tesla (3 T) MRI, Siemens, Germany) with a T1-weighted spin-echo sequence (repetition time (TR) ¼ 150 ms, (echo time) TE ¼ 7.8 ms, voxel size ¼ 0.9 0.9 1, slice ¼ 15, number of excitations (NEX) ¼ 2, resolution ¼ 256). 2.4. In vitro cell culture test of nanoparticles 2.4.1. Human mesenchymal stem cell culture Human mesenchymal stem cells (hMSCs) were isolated from the bone marrow of healthy donors after informed consent. The bone marrow mesenchymal stem
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C.-L. Tseng et al. / Biomaterials 31 (2010) 5427e5435 Triton-X 100 (total lysis group) was adopted as the positive control. After 1, 3, and 7 days of incubation, the culture medium was assayed by the LDH assay kit. In brief, 50 ml of the medium was transferred to a new enzymatic assay plate in which 50 ml of the LDH substrate solution was added. After 30 min, 50 ml of stop solution was added to each well in the plate and read on microplate reader spectrophotometer (SpectraMAX M2; Molecular Devices, USA) at 490 nm optical density (OD). All experiments were repeated 6 times for statistical analysis. 2.5. Nanoparticle cell uptaken study 2.5.1. TEM analysis of cells with internalized nanoparticles TEM was used for the observation of intracellular NPs distribution. Human MSCs were exposed to GdH-NPs suspension for 3 h at 37 C at a concentration of 100 mg/ mL. These hMSCs were washed with PBS 3 times, and then fixed at 4 C in a solution of 2.5% buffered glutaraldehyde for 8 h, then post-fixed in 1% osmium tetraoxide for 2 h, and then washed and dehydrated with increasing concentrations of ethanol and propylene oxide. Cells were embedded in Spurr’s resin and cut into ultrathin sections (50w100 nm). These sections were collected on copper grids and stained with a 1:1 mixture of methanol and lead citrate. The grids were examined using an H-7500 TEM (Hitachi, Japan). 2.5.2. Intracellular Gd accumulation To quantify intracellular Gd concentration, an inductively coupled plasma-mass spectrometer (ICP-MS) (Agilent 7500C, USA) was used in this study. Human MSCs were grown in 10-cm culture dishes. These cells were incubated with GdH-NPs and Gd-DTPA for 12 h at 37 C with a Gd concentration of 20 mg/mL. In control cultures, the cells were placed in a medium without GdH-NPs at the same cell density. Cells were washed 3 times with PBS and then detached by trypsinization. The labeled cell pellets were treated with 65% HNO3 solution at 80 C for 60 min. The samples were diluted to the proper range for analysis. 2.5.3. In vitro MRI of Gd-labeled stem cells To evaluate the GdH-NPs contrast in T1-weighted sequences, cells were incubated with GdH-NPs at concentrations of 10 and 100 mg/mL GdH-NPs for 24 h. Subsequently, cells were trypsinized and washed twice with the culture medium by centrifugation at 1500 rpm for 5 min. After each step the supernatant was harvested again for subsequent MRI examinations to ensure that no GdH-NPs were left in the medium after washing. After the final centrifugation, the cell pellet was analyzed using MRI. MRI was performed using a Magnetom Trio 3-T MRI system with a T1weighted spin-echo sequence (repetition time (TR) ¼ 150 ms, (echo time) TE ¼ 7.8 ms, voxel size ¼ 0.9 0.9 1, slice ¼ 15, number of excitations (NEX) ¼ 2, resolution ¼ 256). To avoid susceptibility artifacts from the surrounding air in the scans, all samples were placed in a water-containing plastic container and evaluated at room temperature. 2.6. Characterization of hMSCs
Fig. 4. The biocompatibility of GdH-NPs for MSCs cultivation examined by the (a) WST-1 and (b) LDH assay with different Gd concentrations after 1, 3, and 7 days of treatment (one-way ANOVA, mean SD, n ¼ 6, *p < 0.05: significantly different). cells (MSCs) were isolated by density gradient centrifugation (1.077 g/cm3, Histopaque) and washed in phosphate buffer saline (PBS). Human MSCs were cultured in alpha-minimum essential medium (a-MEM) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% antibiotic-antimycotic solution, which was the complete a-MEM medium. When the cultures reached 85% confluence, the cells were detached with trypsin-EDTA solution and subcultured at 1 104 cells/mL. 2.4.2. Cell viability Cell proliferation reagent WST-1 kit, was used to examine cell viability. The cells were seeded in 96-well plates at a density of 5 103 cells/well. Cells were incubated with different concentrations of GdH-NPs suspension. The concentrations of GdHNPs were tested at 0, 30, 50,100, and 300 mg/mL. After culturing hMSCs with GdH-NPs for 1, 3, and 7 days, WST-1 reagent was added for cell proliferation assay. After incubation with WST-1 for 2 h at 37 C, the formazan dye generated by the activity of dehydrogenases in cells was proportional to the number of living cells; the absorbance at 420 nm was measured using a microplate reader (SpectraMAX M2; Molecular Devices, USA). All experiments were repeated 6 times for statistical analysis. 2.4.3. Cell toxicity Lysed cells could release lactate dehydrogenase (LDH). Therefore, cell toxicity was evaluated by extracellular LDH content. The LDH was measured by a commercial assay kit (CytoTox 96Ò Assay, Promega, USA). The hMSCs were seeded in 96-well plates at a density of 5 103 cells/well. After a 24-h incubation, the cells were incubated with different concentrations of GdH-NPs (30, 50, 100, and 300 mg/mL). Cells incubated in a medium without GdH-NPs were used as the control group, and
Human MSCs were seeded at a density of 1 106 cells per culture dish (10-cm dishes) and allowed to attach for 24 h. After that, the cells were incubated with GdHNPs (100,10 mg/mL) for an additional 24 h. At the end of incubation, cells were washed 3 times with PBS to remove free GdH-NPs and were harvested using trypsinEDTA. The harvested cells were collected and stained either with fluorescein isothiocyanate (FITC)-conjugated antibodies against human antigens CD29, CD44, CD45,and CD71, or with phycoerythrin (PE)-conjugated antibodies against human antigens CD105 and CD106 (eBioscience Inc, San Diego, CA, USA) [16,19] as described in Table 1. Unlabeled cells are used as a control and isotype antibodies are used, respectively, to exclude nonspecific staining. The cells are incubated at 4 C to diminish the possible nonspecific staining caused by temperature. The incubation should be maintained in the dark to avoid fluorescent attenuation. The cells were examined using flow cytometry (FACSCalibur, Becton Dickinson, USA) to confirm the stem cells’ surface marker. Positive staining was defined as the fluorescence emission that exceeded levels obtained by more than 99% of the cells from the population stained with isotype-matched controls.
3. Results 3.1. Size and morphology of GdH-NPs In this study, the oil-in-water microemulsion system was used to prepare GdH-NPs (Fig. 1). Table 2 shows the size distribution of GdH-NPs prepared in different surfactant (Brij 78) concentrations. Particles size of GdH-NPs prepared by using 4 mM Brij 78 was 138.09 6.90 nm. The particle size at the concentrations of 6, 10, and 30 mM of Brij 78 were 140.17 7.00, 205.70 0.29, and 1131.22 56.56 nm, respectively. There was an enormous increase in the mean particle size from 138.09 nm to 1131.22 nm. The GdH-
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Fig. 5. Surface marker identification of hMSCs (a) before and (b) after GdH-NP cultivation for 24 h at a concentration of 100 mg/mL.
NPs made using 30 mM Brij 78 had 2 major size distributions in the raw data. One was 245.2 45.8 nm and the other was 1459.1 406.4 nm. Because the concentrations of the surfactants are key parameters in preparing NPs by the microemulsion technique, the surface charge of these particles was affected by surfactant concentration. The GdH-NPs made using 4 mM Brij 78 led to the highest zeta potential, 3.8 1.1 mV (Table 2). Other
concentrations of the surfactant affected the zeta potential, producing values that range from 1.6 to þ3.8 mV. Nanoparticles are more stable when the zeta potential is higher. Smaller particle size and higher zeta potential values were observed in the group of the 4 mM Brij 78. The GdH-NPs prepared using the oil-in-water microemulsion template at the concentration of 4 mM Brij 78 was used in the following experiment.
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Fig. 7. Intracellular Gd concentration examined by ICP-MS assay.
3.2. GdH-NPs as an MRI contrast agent Fig. 3(a) shows T1-weighted spin-echo MR images of two kinds of contrast agents, the GdH-NPs, made from microemulsion method, and Gd-DTPA. Both solutions were diluted into different concentrations ranging from 200 to 6.25 mg/mL. The MRI image shows that GdH-NPs produced stronger signal intensity than GdDTPA. The gray-level values of the MRI images are presented in Fig. 3(b). It shows significant differences in gray-level values resulting from GdH-NPs and Gd-DTPA at 50, 100, and 200 mg/mL concentrations. The gray-level value of GdH-NPs at 200 mg/ml was 2.2 times higher than that of Gd-DTPA. 3.3. The biocompatibility of GdH-NPs
Fig. 6. TEM images show the GdH nanoparticles (100 mg/mL, 37 C, and 3 h) internalized into the hMSC cells. (a) Pseudopods were observed at the cell surface (indicated by arrows) and (b) GdH were found in endosomes (indicated by arrows).
Two kinds of electron microscope were used to examine the size and structure of GdH-NPs. Fig. 2(a) was acquired by TEM and Fig. 2 (b) was obtained by SEM. Both figures show that the GdH-NPs were well dispersed and no aggregation was observed. The particle size was about 100 nm. These results are in agreement with the particle size analysis (Table 2). In Fig. 2(a), it can be seen that the central part of the NPs was dark, which may have been caused by Gd encapsulation. SEM equipped with an EDS microprobe analyzer was used to quantify the Gd amount in the NPs. Table 3 provides the results of element analysis corresponding to Fig. 2(b), which illustrates GdH-NPs composted with element of Gd with it. These results indicate that the microemulsion method can be used to prepare nano-scale particles.
To evaluate possible cytotoxic effect of GdH-NPs, cell viability was examined by WST-1 reduction assay. No evidence of cytotoxicity was observed after 1, 3, and 7 days of cultivation with GdH-NPs at concentrations ranging from 30 to 300 mg/mL ((Fig. 4(a)). The viability of hMSCs treated by GdH-NPs at different concentrations was not significantly different from that of the control group (oneway ANOVA, p < 0.05). These data indicated that GdH-NPs were not toxic and were biocompatible for hMSC culture at the tested concentrations. LDH is a stable cytosolic enzyme that is released into the culture supernatant during cell lysis. This may be used to determine the cytotoxicity of cells influenced by different additives or culture conditions. In our study, after incubation with GdH-NPs for 1, 3, and 7 days, the LDH released by cultured hMSCs was collected and measured. The OD value in spontaneous release of LDH from hMSCs with NP treatment was regarded as the control and medium containing Triton X-100 was considered as the positive control (total lysis group). Fig. 4(b) shows that GdH-NPs mediated very low cytotoxicity under concentrations ranging from 30 to 300 mg/mL after co-culture with hMSCs at the indicated times. These results are in agreement with those of the WST-1 test. There was no significant difference between the control and GdH-NPs concentrations ranging from 30 to 300 mg/mL. 3.4. MSC characterization after GdH-NPs co-culture To exclude possible differentiation effects of GdH-NPs on the function of stem cells, we examined the immunophenotypes of labeled hMSCs. As shown in Fig. 5, cell surface antigen profiles of hMSCs were positive for primitive mesenchymal progenitor cell markers, including CD29, CD44, CD71, CD105, and CD 106, whereas
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strong signal intensity change but not the typical bright signal. The MRI image acquired by GdH-NPs (100 mg/mL)-treated group showed a dark image (Fig. 8(b)). 4. Discussion
Fig. 8. Representative T1-weighted MR images of Eppendorf test tubes that contained centrifuged cell pellets after labeling with GdH-NPs in different concentrations for 24 h. (a) hMSCs without labeling, (b) 100 mg/mL and (c) 10 mg/mL GdH-NP labeling.
the hematopoietic lineage-specific marker CD45 was not detected. Other surface markers of hMSCs labeled with GdH-NPs were not affected. Although CD71 and CD106 expression slightly increased after GdH-NPs treatment (11.0 7.94% to 25.5 8.97%; 16.9 12.93% to 30.8 11.51%), no significant difference was observed (One-way ANOVA, mean SD, p < 0.05). 3.5. Intracellular distribution and accumulation of GdH-NPs TEM was carried out to confirm bioadhesion and endocytosis of GdH-NPs with cells. Micrographs of hMSCs incubated with GdHNPs at Gd concentration of 100 mg/mL for 3 h at 37 C are shown in Fig. 6. These micrographs clearly indicated that GdH-NP adhered to hMSCs and the membrane invaginated, forming a pocket containing NPs (Fig. 6(a)). Some particles were incorporated within the cells, and endocytic vesicles (indicated by arrows) with GdH-NPs in the cytosol were also found (Fig. 6(b)). 3.6. Quantification of intracellular Gd concentration Fig. 7 provides the quantitative analysis of the amount of Gd in cells as a contrast agent, with GdH uptake by hMSCs after 24-h cultivation. The results of cells treated with GdH-NPs shows that the Gd concentration in hMSCs was 3.25-fold higher than that in the Gd-DTPA-treated group (3.209 mg/mL versus 1.034 mg/mL, respectively). A one-way ANOVA test showed a significant difference between Gd-DTPA- and GdH-treated groups. The intracellular Gd concentration of hMSCs treated with GdH-NPs was significantly higher than those treated with Gd-DTPA (one-way ANOVA, p < 0.05). 3.7. Cellular MRI imagery We analyzed what concentration of GdH-NPs provided the strongest contrast of hMSCs cells in vitro (Fig. 8(aec)).We found that coincubation of GdH-NPs with hMSCs at 10 mg/mL for 24 h provided the strongest signal of the cell pellet in T1-weighted MRI with bright image (Fig. 8(c))(compared with nonlabeled one, Fig. 8 (a)). Human MSCs cells labeled with 100 mg/mL GdH-NPs showed
For clinical research, transplantation of stem cells for cell therapy needs to be monitored. Labeling of stem cells has become a frequently applied technique to investigate the aforementioned biological properties of stem cells. This study showed that hMSCs incubated with GdH-NPs spontaneously internalized this hydrophobic, nano-sized contrast agent. Cellular uptake after incubation with GdH-NPs was sufficient for the tracking of hMSCs by using T1weighted sequences with clinical MR scanners without toxicity, and would not induce differentiation of hMSCs. MRI has been widely studied as a cell tracking technique in recent years. It has good spatial resolution but in order to become suitable as a cellular imaging modality, its inherently low sensitivity must be dealt with. This may be overcome by using contrast agents with very high relaxivity matter (e.g., by using ferric oxide NPs or a high payload of Gd complexes). In biological tissues, T1 relaxivity is longer than that of T2; therefore, contrast enhancement is better observed with T1-weighted sequences [20]. Moreover, T1-sensitive contrast agents can monitor the distribution of freshly implanted cells more accurately than T2-wighted ferric oxide NP-labeled contrast agents [21]. Gd is the most effective T1 contrast agent for clinical use. Therefore, incorporating Gd3þ into cells for transplantation will change their relaxation characteristics, making it possible to distinguish between labeled and non-labeled cells [22]. Previous studies have shown that lanthanides complexes with dicarbonyl chelating agents have favorable physical properties, thermal stability, and consist of a high weight percentage of the lanthanide metal [23]. In this study, GdCl3, a lanthanides complex, reacted with 3, 4 hexandione (with dicarbonyl compound) to form GdH with hydrophobicity [17]; this is a stable Gd complex that could reduce the free toxic Gd released by unstable Gd-chelate in vivo. Compared to other complexes of Gd (such as Gd-DTPA, a hydrophilic form), GdH with high hydrophobicity could facilitate it with high affinity to phospholipid cell membrane, thus increasing the Gd content in cells [21]. Therefore, GdH-NP was the final NP formulation choice in this study. Methods to enhance MR imaging quality by increasing the imaging ability of contrast agents are as follows: (a) increase the water content in the Gd-complex to increase the water exchanging rate, (b) increase the molecular weight of the Gd-complex to change the Gd- complex relaxivity for increasing the relaxivity rate, and (c) decrease the tumbling rate of the Gd-complex [20]. In this study, the GdH-NPs fabricated by oil-in-water microemulsion method could encapsulate more Gd-chelate in it to form larger Gdcomplexes with high molecular weight causing the slow tumbling rate of GdH-NPs, thus being responsible for the increase in T1 relaxivity that was observed in the strong intensity of the MRI image [2,24]. Therefore, it had better images quality. For stem cell tracking, the safety effect of internalized GdH-NPs should first be investigated. Free Gd ion is more toxic to the human body [2]; therefore, applied Gd-based contrast agents are chelated to form Gd-complex, which increases the biological safety. However, intracellular dechelation of the Gd-DTPA complex liberates the highly toxic Gd ion and causes toxicity. To evaluate possible cytotoxicity effects of GdH-NPs, cell viability and toxicity were examined by WST-1 and LDH assay. Results obtained from these assays (Fig. (4)(a) and (b)) show that the GdH-NPs at the tested concentrations (30 to 300 mg/ml) produced no toxicity. Owing to the result that GdH-NPs at the concentration of 200 mg/ml had good
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T1-weighted signal enhancement from MRI examination (Fig. 3), it could easily be adapted for stem cell labeling. One unexpected outcome of stem cell culture is that typical hMSCs markers such as CD44, CD90, and CD105 can be irrecoverably lost during improper cultivation. After GdH-NPs treatment, the immunotype of hMSCs such as CD29, CD44, CD71, CD105, and CD106 were not affected (Fig. 5) demonstrating hMSCs were not differentiation. Efficient magnetic labeling of cells to be tracked with MRI is of great importance for keeping the detection limit of the cells as low as possible. The uptake of contrast agents by cells can occur via the endocytosis [25]. For endocytosis, the invagination of particles on the cell membrane could forms a vesicle. The hydrophobic property and nano-size of GdH-NPs would facilitate cellular uptake because of the better affinity between GdH-NPs and cell membranes, which enhances transmembrane transport. TEM was utilized to confirm the endocytosis of GdH-NPs (Fig. 6(a), (b)). GdH-NPs were engulfed by cell membrane and entrapped in the endosome. The intracellular Gd accumulation also confirmed that hydrophobic GdH-NPs more easily came into contact with cell membrane and were uptaken into cytoplasm, causing higher Gd accumulation in cells than did Gd-DTPA (Fig. 7). These results agree with a previous study that the amphiphilic structure allows the gadofluorine M molecule to penetrate phospholipid bilayers and to interact with intracellular water protons, which increases R1 relaxation times and leads to the observed increased signal on T1-weighted images [21]. At low concentration of GdH-NPs there will be an increase in signal intensity via T1-weighted signal. From Fig. 3, it shows that GdH-NPs have a superior T1-enhancing ability as compared to that of GdDTPA, and it could improve cellular MRI by using lower Gd concentrations (Fig. 8). Otherwise in Fig. 8(b), hMSCs labeled with 100-mg/ml GdH-NPs caused the T2-weighted image, the dark enhancement. This may have been owing to the high concentration of GdH-NPs labeled on hMSCs; when the concentration of Gd is too high, the effect of T2 relaxation will overcome the effect of T1 and this will suppress the signal of T1 [26]. In this study, we successfully labeled hMSCs with GdH-NPs at a lower Gd concentration but with better T1-weighted MRI images (Fig. 8(c)). Cells that lack substantial phagocytic capacity, such as stem cells, are more difficult to label [21]. One was to use anionic hydrophobic NPs that interact strongly and nonspecifically with the cell plasma membrane because of strong negative surface charges, and easily combine with the lipid cell membrane [27]. The use of hydrophobic molecules, like GdH, might provide a considerable advantage because these molecules can be expected to work with most cell types for endocytosis. Another method to improve hMSCs T1-weighted signal cellular uptake was to couple NPs to antibodies, which penetrate the cell membrane by receptor-mediated internalization [21,28]. Labeling of stem cells via receptor-mediated internalization of molecules might affect the physiological properties of stem cells, such as proliferation, differentiation, or cellecell interactions resulting from the blockade of important receptor-epitopes. Furthermore, it might be problematic if the antibodies will not stick to the NPs surface for a prolonged period of time. 5. Conclusion In this study we successfully synthesized GdH-NPs with promising T1-weighted enhancing properties for MRI applications. The particle size was about 140 nm based on PCS and TEM examination. MRI showed that GdH-NPs have a much higher gray-level signal as compared to Gd-DTPA. From the in vitro cell culture tests, we found that GdH-NPs were biocompatible and were taken up by endocytosis. The intracellular Gd accumulation was much higher in GdHNPs-treated cells than in Gd-DTPA-treated cells. Furthermore,
labeled stem cells showed better signals in cellular MRI assay. The expression of surface antigens of hMSCs after culture with GdH-NPs was examined, and it showed no difference from the control group. We believe that, in the near future, the GdH-NPs developed in this study have great potential as a contrast agent for stem cell tracking by MRI methodology. Appendix Figures with essential color discrimination. Figs. 1 and 5 in this article are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.biomaterials.2010.03.049. References [1] Vuu K, Xie J, McDonald MA, Bernardo M, Hunter F, Zhang Y, et al. Gadoliniumrhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjugate Chem 2005;16:995e9. [2] Mulder WJ, Strijkers GJ, vanTilborg GA, Griffioen AW, Nicolay K. Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed 2006;19:142e64. [3] Pittenger M, Mackay A, Beck S, Jaiswal R, Douglas R, Mosca J, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284 (5411):143e7. [4] Barry F, Murphy J. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36(4):568e84. [5] Chemaly ER, Yoneyama R, Frangioni JV, Hajjar RJ. Tracking stem cells in the cardiovascular system. Trends Cardiovasc Med 2005;15(8):297e302. [6] Hardy J, Edinger M, Bachmann M, Negrin R, Fathman C, Contag C. Bioluminescence imaging of lymphocyte trafficking in vivo. Exp Hematol 2001;29 (12):1353e60. [7] Morawski AM, Lanza GA, Wickine SA. Target contrast agents for magnetic resonance imaging and ultrasound. Curr Opin Biotechnol 2005;16:89e92. [8] Adonai N, Nguyen K, Walsh J, Iyer M, Toyokuni T, Phelps M, et al. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A 2002;99(5):3030e5. [9] Hinds K, Hill J, Shapiro E, Laukkanen M, Silva A, Combs C, et al. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 2003;102(3):867e72. [10] Lanza GM, Winter PM, Caruthers SD, Morawski MA, Schmieder AH, Crowder KC, et al. Magnetic resonance molecular imaging with nanoparticles. J Nucl Cardiol 2004;11(6):733e43. [11] Leslie LC, Nitin N, Gang B. Magnetic nanoparticle probes. Mat Today 2005;8 (5):32e8. [12] Hengerer A, Grimm J. Molecular magnetic resonance imaging. Biomed Imaging Interv J 2006;2(2):e8. [13] Hsiao J-K, Tsai C-P, Chung T-H, Hung Y, Yao M, Liu H-M, et al. Mesoporous silica nanoparticles as a delivery system of gadolinium for effective human stem cell tracking. Small 2008;4:1445e52. [14] Prantner AM, Sharma V, Garbow JR, Piwnica-Worms D. Synthesis and characterization of a Gd-DOTA-D-permeation peptide for magnetic resonance relaxation enhancement of intracellular targets. Mol Imaging 2003;2:333e41. [15] Hill JM, Dick AJ, Raman VK, Thompson RB, Yu ZX, Hinds KA. Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009e14. [16] Croft AP, Przyborski SA. Mesenchymal stem cells from the bone marrow stroma: basic biology and potential for cell therapy. Curr Anaesth Crit Care 2004;15:410e7. [17] Oyewumi M, Mumper R. Engineering tumor-targeted gadolinium hexanedione nanoparticles for potential application in neutron capture therapy. Bioconjug Chem 2002;13(6):1328e35. [18] Mumper RJ, Mills BJA, Ryo UY, Jay M. Polymeric microspheres for radionucide synovectomy containing neutron-activated Holmium-166. J NucI Med 1992;33:398e402. [19] Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: Paradoxes of passaging. Exp Hematol 2004;32:414e25. [20] Tilcock C, Unger E, Cullis P, MacDougall P. Liposomal Gd-DTPA: Preparation and charaterization of relaxivity. Radiology 1989;171:77e80. [21] Nolte IS, Gungor S, Erber R, Plaxina E, Scharf J, Misselwitz B, et al. In vitro labeling of glioma cells with gadofluorine M enhances T1 visibility without affecting glioma cell growth or motility. Magn Res Med 2008;59:1014e20. [22] Modo M, Cash D, Mellodew K, Williams SCR, Fraser SE, Meade TJ, et al. Tracking transplanted stem cell migration using bifunctional, contrast agentenhanced, magnetic resonance imaging. Neuro Image 2002;17:803e11. [23] Mumper RJ, Jay M. The formation and stability of lanthanide complexes and their encapsulation into polymeric microspheres. J Phys Chem 1992;96: 8626e31. [24] Artemov D, Bhujwalla Z, Bulte J. Magnetic resonance imaging of cell surface receptors using targeted contrast agents. Curr Pharm Biotechnol 2004;5(6):485e94.
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