Abstract
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Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis
Abstract
Superparamagnetic iron oxide (SPIO) particles have been used successfully as an intracellular contrast agent for nuclear MRI cell tracking in vivo. We present a method of detecting intracellular SPIO colloid uptake in live cells using cell magnetophoresis, with potential applications in measuring intracellular MRI contrast uptake. The method was evaluated by measuring shifts in mean and distribution of the cell magnetophoretic mobility, and the concomitant changes in population frequency of the magnetically positive cells when compared to the unmanipulated negative control. Seven different transfection agent (TA) -SPIO complexes based on dendrimer, lipid, and polyethylenimine compounds were used as test standards, in combination with 3 different cell types: mesenchymal stem cells, cardiac fibroblasts, and cultured KG-1a hematopoietic stem cells. Transfectol (TRA) -SPIO incubation resulted in the highest frequency of magnetically positive cells (>90%), and Fugene 6 (FUG) -SPIO incubation the lowest, below that when using SPIO alone. A highly regular process of cell magnetophoresis was amenable to intracellular iron mass calculations. The results were consistent in all the cell types studied and with other reports. The cell magnetophoresis depends on the presence of high-spin iron species and is therefore expected to be directly related to the cell MRI contrast level.—Jing, Y., Mal, N., Williams, P. S., Mayorga, M., Penn, M. S., Chalmers, J. J., Zborowski, M. Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis.
The synthetic magnetic nanoparticles possess sufficiently high magnetic moment for applications in remote cell manipulation, including magnetic cell separation (1,2,3,4,5,6,7). Applications of magnetic cell separation include detection of rare cancer cells in circulating blood, detection of fetal cells in maternal blood, and modification of cellular content of the graft for cellular therapies (“graft engineering”) (3, 8,9,10). Other applications include targeted drug delivery (11, 12) and targeted radio-frequency hyperthermia (13) in cancer treatment. In particular, the high magnetic moment of the superparamagnetic iron oxide (SPIO) nanoparticles makes them a suitable candidate for an intracellular, nuclear MRI contrast agent at single-cell resolution (14).
Typically, nanoparticles consist of a polymeric matrix (such as dextran, diameter on the order of 100 nm) with an embedded polycrystalline iron mineral (such as maghemite, diameter on the order of 10 nm) and a surface modified by attachment of functional groups such as amino or carboxyl groups (1, 2, 9, 15). By themselves, the unmodified SPIO particles are inefficient intracellular labels of stem cells or other mammalian cells in vitro (14, 16). However, the addition of a transfection agent with ferumoxides augments synthetic particle translocation to the cytosol and has been shown to facilitate stem-cell labeling efficiency (17, 18).
Transfection agents (TAs) are either positively or negatively charged macromolecules, such as dextrans, phosphates, synthetic lipids, proteins, and dendrimers. Unmodified SPIO nanoparticles bind to TA reagents by electrostatic interactions (19). The biophysical and metabolic properties of stem cells labeled with TA-SPIO complexes have been characterized, and the results show that the intracellular labeling procedure has no effects on cell viability, differentiation, or phenotype (18, 20).
Selection of optimum TA-SPIO complexes is important in order to achieve high-quality MRI cell tracking in the body of the target cell population. It is also important that the TA-SPIO uptake is efficient and uniform across the entire target cell population to ensure accurate MRI localization of all the grafted cells. Recently, a dextran-coated SPIO nanoparticle has been approved by the Food and Drug Administration (FDA) as an MRI contrast agent for hepatic reticuloendothelial cell imaging (Feridex I.V., a ferumoxide-based SPIO nanoparticle) (17).
Unfortunately, current experimental techniques for measuring the intracellular iron uptake and internalization dynamics are limited to fixed specimens. These techniques include Prussian blue staining (21), T2 relaxation measurements by variable-field relaxometer (18), iron content analysis by ferrozine-based spectrophotometric assay (18), and iron atomic absorption spectroscopy (AAS) (22). The only reported method capable of measuring magnetic contrast agent uptake in live cells is MRI imaging; however, its capability is limited by low resolution (18, 21). Moreover, the physicochemical analysis methods available for measuring SPIO uptake by the cell are limited to reporting only mean population value, not single cell values.
Here we propose to measure the single-cell iron uptake by measuring the cell “magnetophoresis,” a term used to characterize the motion of a magnetically susceptible particle in a viscous medium under the influence of an applied magnetic field (23,24,25). The cell magnetophoretic mobility MM [mm3/(T·A·s)] is quantitatively related to the physicochemical properties of the cell-label complex, such as the magnetic susceptibility, number and volume of the magnetic nanoparticles taken up by the cell, the hydrodynamic diameter, and the viscosity of the fluid medium (26, 27). The cell magnetophoresis has been shown to depend on the number of phagocytized iron oxide nanoparticles, and to provide quantitative measure of the amount of internalized iron, in agreement with electron paramagnetic resonance used as a reference method (28).
The cell motion in viscous media is conveniently measured using the principles of particle image velocimetry (PIV) or particle tracking velocimetry (PTV) (29, 30). A specialized method based on a nearly isodynamic magnetic field (characterized by a nearly constant magnetic force acting on the cells) and PTV has been dubbed by us cell tracking velocimetry (CTV, with hundreds to thousands of cells tracked per sample, Fig. 1) (30, 31). We have used this method to measure changes in magnetic susceptibility of single erythrocytes (25, 32), to characterize the cell binding of antibodies conjugated to magnetic nanoparticles (33, 34), and to measure magnetic susceptibility of nanoparticles used for immunomagnetic cell separation (27) and magnetic susceptibility of MRI contrast agent (35). The accuracy and precision of the CTV-tracking algorithm has been validated by measurement of the sedimentation rate of particles of known density and size (36), magnetophoretic motilities of monodisperse, magnetite-coated polystyrene microspheres with known magnetizations (31), and polystyrene microspheres in gadolinium solutions of known susceptibilities (35).
Schematic diagram of cell tracking velocimeter and the region of interest where the magnetic force is nearly independent of position (as indicated by nearly parallel, equal-length cell magnetophoretic tracks, shown as thick lines; the thin lines represent magnetic energy density isolines). The gravitational sedimentation (not shown for clarity) is measured in the direction perpendicular to the direction of cell magnetophoresis.
In this study, we used the cell MM measurements by CTV to determine SPIO uptake by 3 different types of tissue progenitor cells, important for tissue engineering applications: rat mesenchymal stem cell (MSC), rat cardiac fibroblast (CF), and human hematopoietic progenitor cell line KG-1a. The measurements were used to determine the magnitude and distribution of the intracellular iron uptake for different TA and SPIO combinations in an attempt to determine optimal loading parameters for maximizing the accuracy of cell tracking in the body by the MRI.
MATERIALS AND METHODS
Cell culture
Three types of cells were used for investigations. Lewis rat mesenchymal stem cells (MSCs) derived from Lewis rat bone marrow cells (provided by M.S.P.) were cultured at 37°C, 5% CO2 using Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG) (Gibco, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotic and antimycotic (Gibco, Invitrogen) (37,38,39). Rat CFs derived from rat heart (provided by M.S.P.) were cultured at 37°C, 5% CO2 using Iscove’s modified Dulbecco’s medium (Media Core Facility, Cleveland Clinic, Cleveland, OH, USA) supplemented with heat-inactivated 10% FBS and 1% antibiotic and antimycotic (38, 40). KG-1a cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured at 37°C, 5% CO2 using Iscove’s modified Dulbecco’s medium with 20% FBS.
Intracellular iron tagging
Ferumoxide injectable solution (Feridex I.V.; Advanced Magnetics Inc., Cambridge, MA, USA) is a dextran-coated SPIO colloid used for intravenous injection as an MRI contrast medium. It is a nonstoichiometric magnetite mixture of average formula FeO1.44. Each milliliter of ferumoxide injectable solution contains 11.2 mg iron, 5.6–9.1 mg dextran, and 0.25–0.53 mg citrate. The day before transfection, 2 × 105 cells were seeded per well (6-well plate; BD Falcon, Bedford, MA, USA) with 2 ml cell-growth medium. The cells were incubated under their normal growth conditions for 24 h. On the day of intracellular tagging, 9 μl of SPIO and 6 μl of TA (reagents listed in Table 1), both out of bottles as supplied by the manufacturer, at the volume ratio of 3:2, were suspended in 80 μl nonserum, nonantibiotic DMEM medium (Media Core Facility) and mixed by vortexing for 10 s. After incubation for 15 min at room temperature to allow TA-SPIO complex formation, the mixture was added to the cells in a dropwise manner (final SPIO iron concentration in cell medium of 48.5 μg/ml). In experiments using varying iron concentrations, the TA-SPIO solutions were prepared by using larger initial volumes of SPIO and TA (at the same volume ratio of 3:2) followed by 1:2 sequential dilutions before adding the mixture to cells (corresponding to the media iron concentration range between 89.6 and 5.6 μg/ml, inclusive). The cells with the TA-SPIO complex were then incubated for 2 h under their normal growth conditions. After incubation, the medium containing TAs was removed by gentle aspiration and PBS (Media Core Facility) buffer washing.
TABLE 1.
Sources of TAs
| TA | Composition | Vendor |
|---|---|---|
| Superfect (SUP) | Low-generation heat-activated dendrimer | Qiagen, Valencia, CA, USA |
| Lipofectamine 2000 (LIP) | Liposomal agent | Invitrogen Corporation, Carlsbad, CA, USA |
| Transfectol (TRA) | Unknown | GeneChoice, Inc, Frederick, MD, USA |
| Fugene 6 (FUG) | Proprietary blend of lipids and other components supplied in 80% ethanol | Roche Molecular Biochemicals, Indianapolis, IN, USA |
| Exgen 500 (EXG) | 22 kDa linear polyethylenimine | Fermentas Inc., Hanover, MD, USA |
| Targefect F-2 (TAR) | Proprietary blend of nonlipid cationic polymers | Targeting Systems, Santee, CA, USA |
| Peptide enhancer (PEP) | An endosmolytic peptide that enhances the efficiency of targefect-mediated gene transfer in certain cell types. | Targeting Systems, Santee, CA, USA |
Cell magnetophoretic mobility analysis by CTV
The CTV apparatus measures the motion of cells on an individual basis, in a nearly constant magnetic force directed orthogonally to gravity (Fig. 1). The permanent magnet assembly with specially shaped pole pieces produces a magnetic energy gradient Sm (146±1 T·A/mm2), that is nearly constant (isodynamic) in the area where the measurement is made (1.05 mm wide; 0.79 mm high). Consequently, the horizontal cell velocity component induced by the magnetic force stays constant for the time of the measurement, as does the vertical velocity component induced by the gravitational sedimentation. The displacements are observed using a microscope (BXFM-F; Olympus, Tokyo, Japan) with attached 12-bit monochrome video camera (Retiga Exi, QImaging, Burnaby, BC, Canada). The image size is 640 × 480 pixels with 1 × 1 binning. The digital images are acquired to PC RAM using software (Video Savant, IO Industries, London, ON, Canada), and subsequently saved to the PC hard drive. The CTV algorithm uses 5 consecutive frames to establish the most probable paths of cells (n=300 to 700 cells/sample). From this information, the algorithm reports 2D locations. With the aid of Excel (Microsoft Corp., Redmond, WA, USA) spreadsheet macros, a linear fit of location-time data gives the velocity components of each particle. Up to 1500 cells can be measured in 20 min. The final outputs are cell MM and cell sedimentation coefficient s population statistics (including mean, sd, and 95% confidence interval) computed with the aid of additional macros. The cell motion induced by the magnetic field B, through a viscous fluid and opposed by a hydrodynamic drag, is described by the terminal velocity:
where Dc is the cell diameter, η is the viscosity of the suspending fluid, m is the mass of iron, and Ms is the mass saturation magnetization of iron. Magnetophoretic mobility is defined as the ratio of the cell velocity vm induced by a magnetic field to the local magnetic driving force (30), Sm = H dB/dx:
Combination of Eq. 2 and B = μ0H (applicable to free space, where μ0 = 4π × 10−7 T · m/A is the magnetic permeability of free space and H is the local magnetic field strength) allows calculation of the iron mass m of a cell moving with the magnetophoretic mobility MM:
The mass saturation magnetization Ms of the ferumoxides is provided by the manufacturer and is equal to 73 emu/g iron, or 73 × 103 A · m2/kg iron (41). The mean field induction was B = 1.4 T, and the cell diameters Dc were calculated from the cell sedimentation coefficient measured by CTV (see below) and corroborated by Coulter Counter measurements of cell electrical impedance. Due to the lack of operator control over the field magnitude of the permanent magnet in the current experimental setup, the fixed range of the camera acquisition rate, and the limits to the microscope viewing area, there is an upper limit to the magnetically induced cell velocity that can be measured by the CTV. To extend the range of the measurements to high-mobility cells, the viscosity of the fluid medium was increased by the addition of 0.2% methyl cellulose (Sigma-Aldrich, St. Louis, MO, USA; 2% in H2O, viscosity 0.4 Pa·s) in 1× PBS buffer. The final viscosity of the solution was 0.088 Pa · s, measured by laboratory viscometer at room temperature (R.T., in triplicate). The solution used for low-mobility cells was the 1× PBS buffer solution, with a viscosity of 0.01 Pa · s at R.T. Although all the reagents used in this study were from the same lot, the cells used were from different passages of the primary culture, which may have introduced random error to the intracellular iron uptake rate (for example, MSCs used in this study were from passages 13, 15, and 17). Another source of error is cell position discretization; that is, the process of assigning the cell image centroid from among a set of pixels that make up a cell image (35, 36).
Cell size distribution measurement by CTV
With respect to unit gravity sedimentation, the settling velocity of a sphere vs is given by:
where ρc and ρf are the cell density and the suspending fluid density, respectively; g = 9.81 m/s2 is the standard gravity acceleration (or the gravitational field intensity at sea level); and s is the sedimentation coefficient:
The distributions of cell population sedimentation coefficients for all 3 cell types used in this study are shown in Fig. 2. The diameter of a sedimenting cell is calculated by measuring cell sedimentation velocity vs and by combining Eqs. 4 and 5:
The mean density of the MSC, CF, and KG-1a cells are approximated by that of a lymphocyte, 1.065 × 103 kg/m3. The suspending fluid, 1× PBS buffer, has a density of 1.0 × 103 kg/m3 and viscosity of 0.01 Pa · s at R.T. To test self-consistency of the CTV method, the calculated cell-diameter distributions were compared with measured diameter distributions based on cell electrical impedance using Coulter Multisizer II (Beckman-Coulter Corp., Hialeah, FL, USA) with a 70-μm aperture (Fig. 3). The cell diameter distributions determined by these 2 methods were in agreement, with minor random differences. The differences are likely related to the cell density estimation, nonuniform density distribution in the cell population, and the departure from spherical cell shape (assumed in Eq. 6) (42). MSC mean diameter was 12.7 ± 2.1 μm, cardiac fibroblast mean diameter was 11.5 ± 1.6 μm, and KG-1a cell mean diameter was 10.4 ± 1.7 μm.
Cell sedimentation coefficient distributions, as measured by CTV.
Cell diameter Dc distribution as measured by cell electric impedance (Coulter Multisizer, used as a reference) (a), and cell sedimentation coefficient (by CTV) (b). Note agreement between the 2 methods, interpreted as the validation of the CTV tracking algorithm.
Histological analysis
Prussian blue staining was used to demonstrate the presence of ferric iron and ferritin in the cytological preparations of cell suspensions considered for magnetophoretic analysis. Prior to staining, 10% neutral buffered formalin was used to fix the sample. The cells were first stained with a working solution containing equal amounts of 4% potassium ferrocyanide and 4% hydrochloric acid (Polysciences Inc, Warrington, PA, USA). The standard R.T. staining protocol required 2 changes of the solution at 10 min each. After washing with distilled water, the cells were counterstained by nuclear fast red for 4 min and then rinsed in running tap water for 1 min. The microphotographs of the Lewis rat MSCs incubated with different TA-SPIO combinations are shown in Fig. 4. The analysis was used to select those TA-SPIO reagent combinations that provided clear evidence of intracellular iron presence without extracellular contamination.
Microphotographs of Lewis rat MSCs incubated with different TA-SPIO combinations (listed in Table 1) and stained for iron content by Prussian blue: a) negative control, b) SPIO alone, c) SUP-SPIO, d) FUG-SPIO, e) EXG-SPIO, f) LIP-SPIO, g) TAR- SPIO, h) PEP-SPIO, and i) TRA-SPIO. Note evidence of extracellular iron precipitation in panels c, g, and h and of the intracellular iron sequestration in panels f and i. There is no obvious, visual evidence of presence of iron in panels b and d, and there is evidence of marginal extracellular iron precipitation in panel e. As a result, only preparations a, b, d, f, and i were used for cell magnetophoretic analysis.
RESULTS
To guide the choice of the best SPIO and TA combination, Lewis rat MSCs labeled with SPIO alone or with a TA-SPIO complex (at ~50 μg iron/ml medium solution) were stained with Prussian blue and compared to unmanipulated negative controls (Fig. 4). The TA reagents are listed in Table 1. When these cells were incubated with SPIO alone or with Fugene 6 (FUG) -SPIO complex for 2 h, the cellular staining was undetectable to low. The staining was more pronounced for MSCs tagged with Exgen 500 (EXG) -SPIO or Transfectol (TRA) -SPIO complex than with Lipofectamine 2000 (LIP) -SPIO complex. An undesirable particle aggregation and evidence of extracellular iron were observed when MSCs were incubated with Superfect (SUP) -SPIO, peptide enhancer (PEP) -SPIO, and Targefect F-2 (TAR) -SPIO complexes; therefore, these reagents were excluded from studies assessing magnetophoretic mobility measurement by CTV.
Addition of TAs modified with SPIO facilitates iron entry into the cells and presumably increases the MSC magnetic moment during cell culture expansion. The resulting changes in the cell magnetophoresis were measured quantitatively using the CTV apparatus, as described in Materials and Methods. The magnetophoretic mobility distributions were parameterized by the population mean and the magnetically positive cell frequency MM(+). The parameter MM(+)was defined as the fraction of cells whose mobility was higher than the cutoff mobility MMcut, the highest mobility measured for the negative control sample, Fig. 5a [MMcut=4.0×10−5 mm3/(T·A·s)]. Therefore, the cumulative frequency MM(+)represents the fraction of the MSCs that uptake SPIO. Even at a low TA-SPIO concentration in the medium of the test sample (5.6 μg iron/ml), there was an observable increase of
with respect to the negative control, 8.56 × 10−5 vs. 7.12 × 10−6 mm3/(T·A·s), and a large fraction of cells in the test sample moved faster than in the negative control, MM(+) = 0.70 (Fig. 5a).
a) Magnetophoretic mobility MM distribution of the MSCs labeled with LIP-SPIO (test sample) and a negative control (open bars). Maximum mobility of the negative control MMcut is used to define the cell fraction labeled with intracellular iron in the test sample, MM(+), taken as a cell fraction whose mobility is higher than MMcut (as indicated by the broken lines). Also shown are the mean mobility of control and test samples M![[M with macron]](/corehtml/pmc/pmcents/x004Dx0304.gif)
and the number of cells tracked N. b) The fraction of intracellular iron-labeled cells, MM(+), approaches unity at low LIP-SPIO concentration in the media, indicating that the labeling process is efficient (intracellular iron enters most of the cells in the sample). A nearly linear increase of M with LIP-SPIO concentration in solution indicates no constraints to the intracellular iron uptake in the concentration range shown.
The increase in cell MM was dose dependent. The MSCs were incubated with either SPIO alone or under increasing concentrations of the LIP-SPIO complex (prepared by 1:2 sequential dilutions beginning with 89.6 μg iron/ml medium) for 2 h, followed by measurement of the mean MM and the magnetically positive cell fraction (Fig. 5b). The increase of LIP-SPIO complex concentration corresponded to an increase of iron concentration in the incubation medium from 5.6 to 89.6 μg/ml, and resulted in a >20-fold increase of the mean MM of the treated cells, from 7.24 ± 1.99 × 10−5 to 1.67 ± 0.18 × 10−3 mm3/(T·A·s). In this concentration range, the mean MM increased linearly (R2=0.98, each data point in triplicate, corresponding to hundreds of cells tracked). Interestingly, the fraction of the magnetic cells reached a plateau at low iron concentration in the medium, and already MM(+) = 62 ± 8% of cells showed significant iron uptake at 5.6 μg iron/ml medium. This is an indication of an efficient uptake of LIP-SPIO by the entire cell population, an important finding that increases confidence in the use of LIP-SPIO as an intracellular contrast agent for MRI tracking.
The SPIO uptake depended on the type of TA reagent (Fig. 6 and Table 1), and was significantly higher when compared to SPIO alone for LIP-SPIO and TRA-SPIO complexes, but not for the FUG-SPIO complex. The MSC incubation with TRA-SPIO resulted in over 2 orders of magnitude increase in mean MM as compared to incubation with SPIO alone, 4.0 × 10−3 and 3.8 × 10−5 mm3/(T·A·s), respectively. These data suggest an almost complete infiltration of the TRA-SPIO-treated cell preparation [MM(+)=0.98]. We also investigated differences due to cell type in cell MM increase and SPIO uptake when exposed to the same TA-SPIO complexes. The MM was measured for 3 different cell model systems: MSCs (as described above), CF primary cultures, and the KG-1a hematopoietic stem-cell line, at fixed iron concentration in incubation medium (~50 μg/ml as used to prepare the microphotographs in Fig. 4). The iron uptake efficiency determined by the mean MM and magnetically positive cell fraction varied depending on the TA type in a similar manner as that observed for MSCs (Fig. 7). Characteristically, the effect was least pronounced for the KG-1a cell line, whose MM values were lower by an order of magnitude than those of MSCs and CFs. Overall, for all cells, incubation with TRA-SPIO led to the highest fraction of magnetically positive cells (>90%) and that with FUG-SPIO produced the lowest such fraction, below that obtained with SPIO alone.
Different transfection agent-SPIO combinations resulted in differences in MSC intracellular iron uptake, as demonstrated by differences in magnetophoretic mobility distribution histograms for SPIO alone and FUG-SPIO, LIP-SPIO and TRA-SPIO combinations. Broken line indicates MMcut value. Note differences in scale between histograms.
Summary of experiments using different cell types and different TA-SPIO combinations (at 50 μg iron/ml medium). The microphotographs show MSC images following incubation with TA-SPIO reagents; note numerous inclusions of intracellular iron particles for LIP- and TRA-SPIO combinations but fewer inclusions for FUG-SPIO and SPIO alone. The values of M (horizontal bars) and the magnetic cell fraction MM(+) (numbers by the respective horizontal bars) correspond to the amount of intracellular iron inclusions; note high mobility and high magnetic fraction of cells labeled with LIP- and TRA-SPIO (with the exception of MSC, for which MM(+)=0.42). MSC microphotographs are excerpted from Fig. 4.
DISCUSSION
The highly regular cell magnetophoresis process lent itself to quantitative, fluid mechanical analysis of single-cell motion in the magnetic field. A similar approach to the one described here has previously been shown to provide quantitative information about the number of iron oxide nanoparticles phagocytized by mouse macrophages, human dendritic cells, T lymphocytes, and pulmonary epithelial cells, which further strengthens the rationale for this study (28). The calculated amounts of intracellular iron (Eq. 3) were consistent with the published data using other methods (Table 2), such as AAS and T2 relaxation by variable-field relaxometry (17). For example, Frank et al. (18) measured the ferumoxide (25 μg/ml) -Lipofectamine/Plus complex in labeled rat MSCs and reported an iron content of 7.6 pg/cell by ferrozine-based spectrophotometric assay. Using NMR relaxometry, Arbab et al. (21) measured the ferumoxides (50 μg/ml) and protamine sulfate (3 μg/ml) in labeled human MSCs and hematopoietic stem cells (HSCs) and reported an iron content of 10.9 and 2.01 pg/cell, respectively. Considering that cell type, TA type, and the ratio of TA to SPIO are not identical in other studies in the literature, our measurements using MM are similar to those of other groups. That said, an important potential source of error in our calculations is the assumed ferumoxide magnetization value (41). The actual value may be lower because of such factors as nanoparticle oxidation and nonlinear effects of particle aggregation on volume magnetization. In addition, the published iron content data were based on the molecular forms of iron, whereas the iron content calculated from the cell MM is directly related to the magnetic moment of the crystalline intracellular iron, be it γ-Fe2Q3 or Fe3O4. In this sense, the MM data are of greater practical importance because they are directly related to the source of the MRI signal; that is, the magnetic moment of the intracellular iron.
TABLE 2.
Calculated iron mass m (pg/cell)
| Treatment | MSC | CF | KG-1a |
|---|---|---|---|
| SPIO | 0.05 ± 0.01 | 0.28 ± 0.10 | 0.02 ± 0.01 |
| FUG-SPIO | 0.02 ± 0.015 | 0.04 ± 0.01 | 0.01 ± 0.002 |
| LIP-SPIO | 1.40 ± 0.34 | 1.38 ± 0.11 | 0.05 ± 0.01 |
| TRA-SPIO | 5.89 ± 0.79 | 3.71 ± 0.65 | 0.21 ± 0.005 |
Values are means ± sd.
The observed differences in the staining pattern are likely related to the electrostatic interactions between particles and cell structures. Ferumoxides are highly negatively charged SPIO nanoparticles that are difficult to attach to the negatively charged cell membrane without modification of the surface charges. Polycationic TAs bind to the dextran, thereby modifying the distribution of positive and negative surface charges on the ferumoxide surface through electrostatic interactions (19). It has been reported that the efficiency and stability of a TA-SPIO complex (and a TA-DNA complex) will vary according to the surface charge of the macromolecules, concentrations of the nanoparticle and TA, pH of the solution, and time (43). Therefore, it is reasonable to assume that those factors are also the likely reason for the observed variability in the cell magnetophoretic mobility between different TA types. The large amounts of visible magnetic particle aggregation and extracellular precipitation observed for SUP-SPIO, TAR-SPIO and PEP-SPIO complexes are likely related to cross-linking between TAs and multiple ferumoxide crystals, transforming these nanoparticles into large particle clusters, visible under light microscopy (Fig. 4). The lack of evidence of FUG-SPIO particle translocation into the cytoplasm as determined by histochemistry (Fig. 4d) and cell magnetophoresis (Figs. 6b and 7) is in agreement with the published reports on the inefficient iron incorporation in the presence of FUG as measured by NMR relaxometry (44).
In summary, the results obtained by quantitative magnetophoresis of live cells and supplemented by cytochemical staining show significant differences in the intracellular iron uptake between different cell types and TA reagents. A linear increase of the population mean MM with increasing free iron in solution, accompanied by a rapid increase to unity of the magnetic cell fraction indicated efficient iron uptake by all cells (tested on the MSCs). The magnitude of the single-cell MM was consistent with the average amount of the intracellular iron reported by others. Importantly, live cell magnetophoresis is sensitive to the magnetic moment of the intracellular iron, and unlike other methods that measure the concentration of intracellular iron (including the mixture of low-spin and high-spin species), provides information directly applicable to intracellular MRI contrast reagent selection.
Acknowledgments
We thankfully acknowledge support by the U.S. National Institutes of Health (CA62349 to M.Z.) and the National Science Foundation (BES-0124897 to J.J.C.).
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Funding
Funders who supported this work.
NCI NIH HHS (2)
Grant ID: R01 CA062349
Grant ID: CA62349
National Institutes of Health (1)
Grant ID: CA62349
National Science Foundation (1)
Grant ID: BES‐0124897