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Am J Physiol Cell Physiol 293: C1698-C1708, 2007. First published September 26, 2007; doi:10.1152/ajpcell.00215.2007
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VASCULAR BIOLOGY

In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging

Y. Jeffrey Wu,2 Leslie L. Muldoon,2,3 Csanad Varallyay,2 Sheila Markwardt,1 Richard E. Jones,1,2 and Edward A. Neuwelt1,2,4

1Research Service, Veterans Administration Medical Center, and Departments of 2Neurology, 3Cell and Developmental Biology, and 4Neurosurgery, Oregon Health and Sciences University, Portland, Oregon

Submitted 24 May 2007 ; accepted in final form 21 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cellular labeling with ferumoxides (Feridex IV) superparamagnetic iron oxide nanoparticles can be used to monitor cells in vivo by MRI. The objective of this study was to use histology and MRI to evaluate an in vivo, as opposed to in vitro, technique for labeling of mononuclear leukocytes as a means of tracking inflammatory processes in the brain. Long-Evans rats were intravenously injected with 20 mg/kg ferumoxides, ferumoxtran-10, or ferumoxytol with or without protamine sulfate. Leukocytes and splenocytes were evaluated by cell sorting and iron histochemistry or were implanted into the brain for MRI. Injection of ferumoxides/protamine sulfate complex IV resulted in iron labeling of leukocytes (ranging from 7.4 ± 0.5% to 12.5 ± 0.9% with average 9.2 ± 0.8%) compared with ferumoxides (ranging from 3.9 ± 0.4% to 6.3 ± 0.5% with average 5.0 ± 0.5%) or protamine sulfate alone (ranging from 0% to 0.9 ± 0.7% with average 0.3 ± 0.3%). Cell sorting analysis indicated that iron-labeled cells were enriched for cell types positive for the myelomonocytic marker (CD11b/c) and the B lymphocyte marker (CD45RA) and depleted in the T cell marker (CD3). Neither ferumoxtran-10 nor ferumoxytol with protamine sulfate labeled leukocytes. In vivo ferumoxides/protamine sulfate-loaded leukocytes and splenocytes were detected by MRI after intracerebral injection. Ferumoxides/protamine complex labeled CD45RA-positive and CD11b/c-positive leukocytes in vivo without immediate toxicity. The dose of feumoxides in this report is much higher than the approved human dose, so additional animal studies are required before this approach could be translated to the clinic. These results might provide useful information for monitoring leukocyte trafficking into the brain.

leukocyte trafficking; brain; B lymphocyte; rat

SUPERPARAMAGNETIC IRON OXIDE (SPIO) and ultra-small SPIO (USPIO) nanoparticle agents, consisting of an iron oxide core with a variable carbohydrate coating, are gaining use for MRI. Ferumoxides (Feridex IV), a dextran-coated SPIO nanoparticle (hydrodynamic size: 120–180 nm), is rapidly opsonized and endocytosed (21, 39). Ferumoxides are approved by the United States Food and Drug Administration (FDA) as a MRI contrast agent. USPIO nanoparticles, such as the investigational agents ferumoxtran-10 and ferumoxytol, have particle diameters of 20–50 nm and have longer plasma half-lives of 14–30 h (21, 44). Although most commonly used for liver and lymph node imaging (5, 6, 9, 17, 25, 39), USPIO agents also show excellent potential for imaging intracerebral tumors and neurological lesions in the central nervous system (CNS) (19, 30, 41). Ferumoxides have not been effective for CNS imaging (19, 30, 41). These agents are commonly used at higher doses in animal studies than in clinical trials, because of differences in clearance half-lives and biodistribution, but results have been similar to low-dose studies in humans.

Labeling of cells with SPIO nanoparticles can be used to monitor the migration, biodistribution, and behavior of cells in vivo with MRI. When used alone, ferumoxides do not efficiently label nonphagocytic or nonrapidly dividing mammalian cells in vitro (4). Complex formation between ferumoxides and a variety of transfection agents occurs through electrostatic interactions and enhances iron uptake in multiple cell types. Arbab et al. (3, 4) have shown that complexing ferumoxides with the low-molecular-weight cationic peptide protamine sulfate can enhance cell labeling in vitro compared with ferumoxides alone. Although not as efficient as lipid transfection agents, protamine sulfate has the advantage of being approved by the FDA for human use as a heparin antagonist. The ferumoxides-protamine sulfate (Fe-Pro) complex is rapidly internalized into endosomes and/or lysosomes in vitro (3, 4). The cellular uptake, biodistribution, and half-life of SPIO and USPIO agents is determined by size, coating, and charge (12), all of which may be affected by complexing with protamine. Efficient iron labeling is well tolerated in vitro and does not induce apoptosis, inhibit differentiation, or block cell migration in vivo (2–4). In vitro labeled cells have been used for in vivo MRI tracking and localization studies after implantation or intravenous infusion in animals (2, 5, 13, 46) and humans (14, 47). Iron oxide-labeled cells appear as hypointense areas in tissues on iron-sensitive T2 and T2*-weighted images (8).

The purpose of this study was to explore whether ferumoxides complexed to protamine sulfate could be used for in vivo labeling of leukocytes following intravenous injection. We hypothesized that such an approach would improve cellular labeling in vivo compared with SPIO or USPIO agents alone and would avoid the extensive manipulations associated with in vitro ferumoxides labeling. We evaluated the iron labeling efficiency and toxicity of the protamine complex method for in vivo labeling using different iron nanoparticle magnetic resonance (MR) contrast agents (ferumoxides, ferumoxtran-10, and ferumoxytol). Finally, we characterized the phenotype of in vivo Fe-Pro complex-labeled leukocytes and splenocytes by flow cytometry and imaging properties of these iron-labeled cells in the brain by MRI. A potential limitation of this study is that the dose (20 mg/kg) used for ferumoxides in the rat is much higher than the approved dose (0.56 mg/kg) in humans; nonetheless, the present study suggests that additional animal studies are warranted.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Ferumoxides (Feridex IV), ferumoxtran-10 (Combidex), and ferumoxytol were all supplied by Advanced Magnetics (Cambridge, MA). Protamine sulfate from American Pharmaceuticals Partner was purchased through Ben Venue Labs (Bedford, OH).

In vitro iron labeling and quantification. Isolated cells (2–10 x 106 cells/ml) were mixed with Fe-Pro complex at a final concentration of 50 µg/ml ferumoxides and 5 µg/ml protamine sulfate for 2 h, as described by Arbab et al. (3), or with equivalent preparations of USPIO agents ferumoxtran-10 or ferumoxytol. Iron content within in vitro labeled cells was evaluated using the Quantichrom iron assay (BioAssay Systems, Hayward, CA) with the manufacturer's protocol. Briefly, 50 µl of standards or samples containing 106 cells were mixed with 200 µl Quantichrom Working Reagent in a 96-well plate (in triplicate) and incubated at room temperature overnight. The iron concentration in experimental samples was determined by comparison of the optical density at 565 nm with the standard curve.

In vivo iron labeling. The care and use of animals was approved by the Institutional Animal Care and Use Committee and was under the supervision of the Oregon Health and Sciences University Department of Animal Care. Fe-Pro complex was prepared by mixing ferumoxides and protamine sulfate [10:1 (wt/wt) ratio] in sterile saline for 10–15 min with gentle intermittent shaking. Ferumoxtran-10 and ferumoxytol were treated similarly. Normal Long-Evans rats (female, 220–250 g) received Fe-Pro complex or another iron nanoparticle complex by intravenous injection into the femoral vein at 20 mg/kg final iron concentration. Although this dose is higher than the approved human dose of 0.56 mg/kg, it is consistent with previous animal studies (28). For the cell type characterization experiments, the experimental groups consisted of 1) Fe-Pro complex (n = 12), 2) ferumoxides alone (n = 6), 3) ferumoxtran-10-protamine sulfate (n = 4), and 4) ferumoxytol-protamine sulfate (n = 4). Protamine sulfate (2 mg/kg) was used as a control treatment (n = 8). Two and twenty-four hours after injection, whole blood and spleens were collected for leukocyte and splenocyte isolation. Tissue samples from the lung, liver, kidney, spleen, and brain were dissected and fixed in 10% formalin for assessing iron biodistribution.

Cell isolation and phenotype analysis. Mononuclear leukocytes were isolated from circulating blood using LSM Ficoll Lymphocyte Separation Medium (MP Biomedicals, Aurora, OH) and centrifugation following the manufacturer's procedures. Briefly, anticoagulant-treated blood diluted with saline [1:1 (vol/vol)] was layered over separation medium and centrifuged at 400 g for 30 min. Mononuclear leukocytes were recovered from the buffer coat layer (plasma-leukocyte separation medium interface) after centrifugation and washed for future use.

To form a single-cell suspension of splenocytes, the spleen was first pressed through a fine mesh screen. The resulting single-cell suspension was treated with Red Cell Lysis Buffer (Sigma, St. Louis, MO) and washed by centrifugation as previously described (20). We investigated splenocytes because the spleen is a major leukocyte reservoir in the body and large numbers of cells can be isolated for phenotypic analysis. CD45RA-positive (B lymphocytes) and CD11b/c-positive (myelomonocytic) cells were sorted from splenocytes using a selection procedure with monoclonal antibodies specific for rat CD45RA and CD11b/c (Pharmingen, San Diego, CA). Splenocytes were incubated first with anti-rat CD45RA antibody for 20 min at 4°C, washed by centrifugation, and similarly incubated with goat anti-mouse IgG magnetic beads (Miltenyi, Auburn, CA). Splenocytes were then applied to a magnetized column (Miltenyi). The magnetized column was rinsed, and CD45RA-depleted cells were collected with the flow-through supernatant. CD45RA-enriched cells were rinsed from the column after demagnetization. CD45RA-negative (flow through) aliquots were further incubated with CD11b/c antibody by repeating the procedure for enrichment or depletion. Aliquots of the splenocyte subpopulations were collected and subjected to iron histochemistry or flow cytometric analysis to confirm the cell phenotype as previously described (20). The specific florescent antibodies used in flow cytometric analysis were directly coupled to phycoerythrin or FITC. Splenocytes were incubated first with unlabeled isotype-matched nonspecific antibody for 10 min at 4°C (Pharmingen), subsequently incubated with fluorescent-labeled specific antibody for 15 min at 4°C (Pharmingen), washed, and resuspended in propidium iodide (2 µg/ml, Sigma). Labeled cells were then analyzed by FACscan (Becton Dickinson, Franklin Lakes, NJ) using a live cell gate (FL3 low events, on FL2 vs. FL3 dot plot).

Histology and immunocytochemistry. Cells were fixed in 10% (wt/vol) formalin for 15–30 min at room temperature before being blocked in 0.25% (vol/vol) normal goat serum for 30 min at 20°C. Primary antibodies diluted in blocking buffer (1:200 dilution) were added for 16 h at 4°C. Mouse anti-rat CD45RA, CD11b/c, CD3, and CD71 monoclonal antibodies were purchased from Serotec (Raleigh, NC). Mouse anti-rat CD68 monoclonal antibody was purchased from BD Pharmingen (San Diego, CA). After a wash step, cells were incubated for 2 h at 20°C in goat anti-mouse IgG1-Alexa 529 (Molecular Probes, Eugene, OR), each diluted to 1:1,000 in blocking buffer. Cell nuclei were counterstained with Hoechst 3342 (1 µg/ml, Sigma). The signal was observed under a fluorescent microscope with the proper filter. Pathology on rat tissue samples was performed by the ARUP Laboratory (Salt Lake City, UT). For blood count analysis, 0.5 ml of whole blood collected in EDTA microtubes were analyzed in duplicate on a Hemavet 850 (CDC Technologies, Oxford, CT). Iron histochemistry was performed using Prussian blue staining with or without diaminobenzidine (DAB) enhancement (29). Cells or tissues were fixed, washed, and incubated for 20–30 min with 2% potassium ferrocyanide in 3.7% hydrochloric acid, followed by an incubation in hydrogen peroxide-activated DAB solution for 5–10 min. Fe-Pro complex labeling efficiency was determined by manual counting of iron stained and unstained cells using a Zeiss microscope (Axioplan Imaging II, Zeiss, Oberkochen, Germany) and Axiovision 4 software (Zeiss). Images were processed by Adobe Photoshop 7.0 (San Jose, CA). The percentage of labeled cells was determined from the average count of 5–10 high-powered fields (100–400 cells).

MRI. MRI was performed on a Siemens Magnetom Trio 3T scanner using a custom rat head coil as previously described (29). T1, T2, and T2*-weighted sequences were acquired with the following parameters: coronal T1 weighted spin echo, repetition time(TR)/echo time(TE): 750 ms/12 ms, field of view (FOV): 25 x 50 mm, matrix: 96 x 192, slice thickness: 2 mm with a 0.4-mm interslice gap; coronal T2 weighted turbo spin echo scans, TR/TE: 5430 ms/78 ms, echo train length: 13, FOV: 25 x 50 mm, matrix: 96 x 192, slice thickness: 2 mm with a 0.4-mm interslice gap; and T2* weighted gradient recalled echo 3D, TR/TE/flip angle: 21 ms/6.1 ms/25°, FOV: 50 x 50 mm, matrix: 256 x 256, and slice thickness: 2.4 mm contiguous slices in the coronal and 1.7 mm contiguous slices in the axial plane.

For in vitro MR assessment, mononuclear leukocytes, plasma, and whole blood were isolated from rats 2 h after an intravenous injection with Fe-Pro complex, ferumoxytol (20 mg/kg) + protamine sulfate (2 mg/kg), or protamine alone following the procedure described above. Aliquots (150 µl) containing of 108 mononuclear leukocytes, plasma, or whole blood were mixed with 0.5% agarose (1:1 ratio), transferred to a 96-well plate, and rapidly cooled on ice. The mixture became a homogenous solid and was then subjected to MR analysis.

For the in vivo MRI, female Long-Evans rats (220–250 g) were inoculated with Fe-Pro complex or protamine alone, and peripheral leukocytes (n = 8) and splenocytes (n = 6) were isolated as described above. Leukocytes (1.2–2.1 x 106 cells in 24 µl) or splenocytes (1.2–2.1 x 106 cells in 24 µl) were isolated as described above. Using a Hamilton syringe, cells were injected at stereotactic coordinates for intracerebral localization in the right and left caudate putamen (vertical, bregma –6.5 mm; lateral, bregma ± 3.1 mm) as previously described (36). MRI was performed at 2 h and 2 days after intracerebral inoculation.

After MRI, brains were excised from euthanized rats and fixed in formalin. Vibratome sections (100 µm) were assessed for histochemical analysis with hematoxylin staining and DAB-enhanced iron staining to confirm iron localization as previously described (28) (data not shown).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In vitro iron labeling and quantification. Ferumoxides alone labeled 20–60% of cells (depending on the experiment), whereas FePro commonly labeled near 100% and staining intensity was significantly elevated. USPIO agents ferumoxtran-10 and ferumoxytol labeled 1–5% of cells in vitro, and this was not significantly enhanced by the inclusion of protamine. Using a colorimetric iron assay, we found that the iron content per cell increased from 0.32 pg (protamine only) to 0.84 pg (ferumoxides) and 26.0 pg (Fe-Pro complex) 2 h after cells had been labeled. We observed that Fe-Pro complex easily passed through a 30-µm filter without altering in vitro labeling, filtration at 5 µm reduced cell labeling by ~50%, and the complex was completely removed by passage through a 0.2-µm filter (data not shown).

In vivo mononuclear leukocyte iron labeling and cytotoxicity. Ferumoxides premixed with protamine sulfate enhanced in vivo iron labeling of mononuclear leukocytes after intravenous injection. The percentage of labeled cells was variable between different experiments, but protamine generally resulted in a doubling of labeled leukocytes. Intravenous injection of 20 mg/kg FE-Pro complex resulted in iron labeling of leukocytes (ranging from 7.4 ± 0.5% to 12.5 ± 0.9% with average 9.2 ± 0.8%) compared with ferumoxides (ranging from 3.9 ± 0.4% to 6.3 ± 0.5% with average 5.0 ± 0.5%) or protamine sulfate alone (ranging from 0% to 0.9 ± 0.7% with average 0.3 ± 0.3%) (Fig. 1). In contrast to ferumoxides, USPIO iron nanoparticle agents ferumoxtran-10 or ferumoxytol with protamine sulfate did not iron label rat leukocytes in vivo. We observed that FE-Pro complex but not protamine sulfate alone increased the density of leukocytes (buffy coat) as determined by their sedimentation following centrifugation through a Ficoll lymphocyte separation medium. The percentage of mononuclear leukocytes labeled by Fe-Pro complex dropped from 9.2 ± 0.8% at 2 h after injection to 5.9 ± 0.7% at the 24-h time point (Fig. 1C), suggesting that the optimal time required for maximal iron labeling is likely to occur within this time frame. The brown color of plasma in animals given ferumoxtran-10 + protamine and ferumoxytol + protamine, but not Fe-Pro complex or ferumoxides alone, suggested that both ferumoxtran-10 and ferumoxytol are extracellular but ferumoxides is intracellular at the 2-h time point.


Figure 1
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  		Fig. 1. In vivo iron labeling of rat mononuclear cells with ferumoxides in the presence of protamine sulfate (Pro). Female Long-Evans rats received intravenous (IV) ferumoxides, ferumoxtran-10, or ferumoxytol (20 mg/kg) with or without preincubation with Pro (2 mg/kg). A: mononuclear cells were isolated from the peripheral circulation 2 h after injection and stained with diaminobenzidine (DAB)-enhanced Perl's blue. Iron-labeled cells with brown stain are indicated by arrowheads. B: percentages of iron-positive stained cells in the total mononuclear cell population. C: time course of ferumoxides-Pro (Fe-Pro) in vivo iron-labeled rat mononuclear cells. Data are represented as means ± SE.

 
We investigated if FE-Pro complex is toxic to the animals after intravenous injection. Rats showed no acute toxicity or weight loss. FE-Pro complex increased the percentage of circulating neutrophils from 28.7 ± 1.1% to 47.4 ± 1.1% and decreased the percentage of lymphocytes from 65.3 ± 2.9% to 47.8 ± 1.8% at 2 h after injection, although the total white blood cell number was not affected by each treatment. These changes were reproducible, and all values were within the wide normal range in these rats. This effect was not observed in either ferumoxtran-10 + protamine-treated or ferumoxytol + protamine-treated rats. The reasons for the changes of neutrophil and lymphocyte percentages by FE-Pro complex are unknown and need to be further investigated.

We assessed the tissue distribution of iron after administration of the nanoparticle agents with or without protamine. Iron nanoparticles were found in both the kidney and liver after injection of either Fe-Pro or ferumoxtran-10 + protamine versus liver but not kidney localization after ferumoxytol + protamine or ferumoxides alone. The iron nanoparticles were engulfed in Kupffer cells in the liver in all iron agent-treated rats (Fig. 2). Iron nanoparticles were localized in collecting tubules of the renal tissue in Fe-Pro complex- and ferumoxtran-10 + protamine-treated rats. The reasons for the differences in iron tissue distribution caused by different iron agents are unclear. Finally, there is no evidence that any iron nanoparticle caused any significant toxicity in the lung, liver, and kidney of these animals according to pathology reports.


Figure 2
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  		Fig. 2. Biodistribution of iron nanoparticles in tissues of rat after intravenous injection in the presence or absence of Pro. Female Long-Evans rats received intravenous ferumoxides, ferumoxtran-10, or ferumoxytol (20 mg/kg) with or without preincubation with Pro (2 mg/kg). The liver and kidney were collected and fixed in 10% formalin. Tissues were stained with Prussian blue for iron localization in combination with hematoxylin and eosin. All images are x200 magnification.

 
Characterization of in vivo iron-labeled cells. We evaluated the degree to which in vivo iron labeling influenced the relative numbers of monocytes and lymphocytes in the spleen. Spleen cells from rats injected with intravenous Fe-Pro complex were compared with control rats injected with protamine alone. We found ~7.4% of splenocytes were iron stained positive following intravenous Fe-Pro complex injection compared with 0.8% following protamine sulfate alone (Fig. 3A). Compared with control animals, intravenous Fe-Pro complex may have slightly altered the relative percentages of CD11b/c-positive (myelomonocytic) spleen cells (11.1% vs. 18.0%; Fig. 3B). No remarkable effects were noted on CD45RA-positive (B cells; 19.0% vs. 16.4%) and CD3-positive T cell subpopulations (43.3% vs. 43.3%; Fig. 3, B and C). Thus, the overall cell distribution pattern was affected only slightly by the Fe-Pro complex treatment.


Figure 3
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  		Fig. 3. Phenotyping of different cell antigens of splenocytes from rats injected with intravenous Pro or FE-Pro complex. Long-Evans rats received intravenous ferumoxides (20 mg/kg) with preincubation with Pro (2 mg/kg). Splenocytes were collected and subjected to magnetic column sorting for CD11 b/c. A–C: comparisons between control and Fe-Pro complex-treated rat splenocytes. D–F: comparisons between CD45RA-depleted and -enriched splenocytes from a Fe-Pro complex-treated rat. G–I: comparisons between CD11b/c-depleted and -enriched splenocytes from the CD45RA-depleted population of a Fe-Pro complex-treated rat. A, D, and G: percentages of iron-stained positive splenocytes detected by DAB-enhanced Perl's stain. Data are presented as means ± SE. B, C, E, F, H, and I: flow cytometry percentages of specific cell populations using the different surface antigen antibodies indicated in the x- and y-axes.

 
To determine which cell types were in vivo labeled with ferumoxides nanoparticles, we used magnetic column separation to enrich/deplete spleen cells based on the expression of CD45RA (B cells) and CD11b/c (monocytic cells). Ferumoxides uptake in vivo was associated with the expression of CD45RA, since 84.6% of CD45RA-enriched splenocytes were iron stain positive compared with only 12.0% iron stain-positive cells following depletion (<1%) of CD45RA-positive cells (Fig. 3, D and E). Detection of iron labeling was not due to the magnetic beads used in the fractionation procedure since <10% of CD45RA-enriched spleen cells from control (protamine alone) donors possessed detectable iron (data not shown). Furthermore, enrichment was not simply due the interaction between in vivo iron-labeled cells and the magnetic column since column enrichment was <10% of cells from in vivo injected Fe-Pro complex donors in the absence of magnetic beads (data not shown). Ferumoxides uptake was not associated with the expression of CD3-positive T cells since CD45RA cells (enriched for B lymphocytes) possessed mostly iron-labeled cells but very few (<2%) CD3-positive T lymphocytes and CD45RA-depleted cells possessed many (>66%) CD3-positive cells (Fig. 3F) with only 12% iron labeling. CD45RA-depleted cells were further depleted or enriched for cells expressing CD11b/c. Ferumoxides uptake was associated with the expression of CD11b/c but not with the expression of CD3 (Fig. 3, G–I). Thus, the percentage of iron-stained splenocytes was positively correlated with the percentage of CD45RA- and CD11b/c-positive cells and negatively correlated with CD3-positive cells. Using immunohistochemical staining, a comparison between CD11b/c-enriched versus -depleted spleen cells from Fe-Pro complex-treated donors further demonstrated an association between iron staining and expression of CD11b/c and CD68, whereas the absence of iron staining occurred in the population with high CD3 expression (Fig. 4). These data agreed with our flow cytometric data. In addition, we found that CD71 (transferrin receptor) was weakly expressed in the cytoplasm of CD11b/c-positive cells compared with the CD11b/c-negative cell population (Fig. 5). These results suggested that spleen cells expressing CD45RA (B cells), CD11b/c (myelomonocytic lineage), and CD68 (monocyte/macrophage/dendritic cells), but not CD3 (T cells), are involved in the cellular uptake of Fe-Pro complex following intravenous particle administration. Furthermore, strong expression of CD71 was not associated with iron uptake by CD11b/c-positive cells.


Figure 4
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  		Fig. 4. Histochemical analysis of CD11b/ c-depleted and -enriched populations of splenocytes from rats injected with intravenous FE-Pro complex. Long-Evans rats received intravenous ferumoxides (20 mg/kg) with preincubation with Pro (2 mg/kg). Splenocytes were collected and subjected to magnetic column sorting for CD11 b/c. Splenocytes were characterized by immunohistochemistry using different antibodies for CD11b/c, CD3, and CD68. Nuclei stained by Hoechst for each represented field are indicated in the insets of each micrograph.

 

Figure 5
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  		Fig. 5. Fe-Pro complex-induced transferrin receptor (CD71) translocation in splenocytes. Spenocytes were isolated from rats 2 h after intravenous injection of Pro in the presence of ferumoxides. After the CD11b/c magnetic sorting, cells were fixed and stained with CD71.

 
MRI assessment of iron-labeled cells. We tested whether in vivo iron-labeled mononuclear leukocytes could be visualized by MRI. Whole blood, plasma, and mononuclear leukocytes were obtained from rats intravenously injected with ferumoxytol + protamine, Fe-Pro complex, or protamine alone (iron concentration of 20 mg/kg and protamine sulfate of 2 mg/kg). Samples in agarose gel were MR imaged in a 96-well microtiter plate. The presence of iron is shown by the T2 shortening effect (signal loss) in Fig. 6. In samples from animals given ferumoxytol + protamine, the iron signal was found in plasma and whole blood but not isolated cells, indicating that ferumoxytol + protamine is not taken up into cells. Samples from animals given protamine alone showed no iron signal in plasma or leukocytes, whereas whole blood demonstrated noticeable T2 shortening from red blood cell hemoglobin. MRI detected iron in the well containing isolated leukocytes after FE-Pro complex, whereas no T2 shortening was detected in plasma, indicating that ferumoxides were completely cleared from the plasma. Whole blood from Fe-Pro complex-treated animals showed clearly more iron signal than the control protamine blood sample (Fig. 6).


Figure 6
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  		Fig. 6. In vitro T2-weighted MRI of in vivo iron-labeled cells. Female Long-Evans rats received intravenous Fe-Pro complex, ferumoxytol (20 mg/kg) + Pro (2 mg/kg), or Pro alone. Whole blood, plasma, and mononuclear leukocytes were isolated after 2 h and transferred to a 96-well microtiter plate in 0.25% agarose. The presence of iron is indicated by a T2 shortening effect (signal loss). Iron particles can be identified in the well containing isolated leukocytes after in vivo iron labeling with FE-Pro complex but not after ferumoxtyol-Pro injection.

 
Finally, we examined if we could monitor in vivo iron-labeled mononuclear leukocytes and splenocytes in rat brains by MRI. In vivo Fe-Pro complex-loaded leukocytes from peripheral blood (1.2–2.1 x 106 cells total; ~1.0–1.5 x 105 iron-labeled cells) and splenocytes (1.2–2.1 x 106 cells total; ~1.0–1.5 x 105 iron-labeled cells) were intracerebrally injected in the rat brain. An equal quantity of cells (mononuclear leukocytes and splenocytes) from rats given intravenous protamine sulfate alone was injected into the opposite hemisphere of the same recipient animal as a control. Immediately (within 2 h) after intracerebral injection of cells, the MR signal (T1, T2, and T2* weighted) from in vivo iron-labeled mononuclear leukocytes (Fig. 7A, right hemisphere) primarily present along the white matter tract was stronger than control unlabeled leukocytes from protamine sulfate alone-treated animals (Fig. 7A, left hemisphere) within the same animal. There were very slightly hypointense regions along the needle track in the left control hemisphere. However, MR signal changes progressively faded within 2 days following implantation. Similar results were found with splenocyte injection (Fig. 7B). Splenocytes showed stronger hypointense MR signal than leukocytes from matched animals, and the signal appeared to last longer, perhaps due to the higher initial signal strength. The stronger MR signal in splenocytes correlated with more intense iron staining of these cells, likely due to higher Fe-Pro complex iron uptake.


Figure 7
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  		Fig. 7. Fe-Pro in vivo labeled mononuclear leukocytes and splenocytes can be monitored by MRI. Mononuclear leukocytes and splenocytes were isolated from 2 normal rats 2 h after intravenous injection of Pro in the absence or presence of ferumoxides (20 mg/kg). Mononuclear leukocytes (1.2–2.1 x 106 cells; A) and splenocytes (1.2–2.1 x 106 cells; B) from Pro- and FE-Pro complex-treated rats were intracerebrally injected to the left (L) and right (R) hemispheres of the brain, respectively. Rats were subjected to serial MR scans within 2 h (day 0) and 48 h after inoculation (day 2). Arrows indicate signal changes primarily present along the white matter tract. The arrowhead indicate the needle track.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we investigated the effects of protamine sulfate on in vivo cellular iron labeling, tissue biodistribution, and toxicity for SPIO MR contrast agents. Our goal was to improve cellular labeling in vivo compared with SPIO or USPIO agents given alone intravenously while avoiding the extensive manipulations associated with in vitro ferumoxides labeling. We have shown, for the first time, that intravenous administration of Fe-Pro complex SPIO but not ferumoxtran-10 and ferumoxytol USPIO nanoparticles enhances superparamagnetic iron labeling of circulating mononuclear leukocytes.

Oude Engberink et al. (32) reported that SPIO but not USPIO in vitro iron labeled human primary monocytes isolated from peripheral blood. In rat experimental autoimmune encephalomyelitis models, Brochet et al. (7) found that MRI after USPIO (300 µM Fe/kg) intravenous injection allows in vivo visualization of the presence of macrophages in the CNS. They proposed mechanisms involved that either the active incorporation of USPIO nanoparticles in the peripheral blood and transport in the CNS by macrophages or their uptake across the open blood-brain barrier close to the intraparenchymal inflammatory sites or both. In humans with multiple sclerosis, Dousset et al. (15) found that intravenous injection of USPIO at dose of 2.6 mg/kg enhanced lesions on high signal intensities on T1-weighted images and low signal intensities on T2-weighted images by MRI.

Several characteristics of the different nanoparticles used in this study may explain the differences in cell and tissue uptake. Cellular uptake is affected by particle size, coatings, and charge. Matuszewski et al.(26) found that larger carbodextran-coated SPIOs resulted in improved cellular uptake in vitro, whereas Metz et al.(27) found that SPIO surface properties rather than particle sizes may have larger impacts on monocyte phagocytosis. The polycationic protamine sulfate binds to the dextran coat of the iron particles through electrostatic interactions, thereby modifying the distribution of positive and negative surface charges that can adhere to the cell membrane (23). The uptake of negatively charged particles is mediated by scavenger receptors expressed in both Kupffer and endothelial cells (34, 43). Blinded pathological review of our biodistribtuion study showed that uptake of SPIO and both USPIO particles with the dextran coating (ferumoxtran-10) or with a synthetic carbohydrate coating (ferumoxytol) was associated only with Kupffer cells in the liver, similar to previously published results (22). USPIO liver distribution was unaffected by protamine, whereas renal collecting tubular distribution was altered by protamine. The difference in renal distribution between ferumoxtran-10 + protamine and ferumoxytol + protamine might be due to the nonionic coating in ferumoxan-10 versus anionic coating in ferumoxytol, differences in macrophage phagocytosis (45) or blood pool clearance (40, 45). The present study was not designed to investigate further differences between agents.

Our experiments demonstrated which specific cell types might be involved in cellular iron nanoparticle uptake after intravenous administration of FE-Pro complex. Thus, cells expressing CD45RA or CD11b/c along with CD68 but not CD3 may play an important role in FE-Pro complex uptake. B cells (CD45RA), macrophages (CD68), and dendritic cells (CD11b/c) are each capable of phagocytic activity prior to antigen presentation (16, 31, 38). Lane et al.(24) proposed that CD4-positive, CD3-negative cells are involved in both organizing lymphoid tissue and supporting T cell help for B cells in memory antibody responses. SPIOs are efficiently internalized into macrophages and other phagocytic cells after intravenous injection (11). Trehin et al. (42) also found the multimodal nanoparticle CLIO-Cy5.5 was localized in CD11b-positive cells when given through the tail vein in a mouse brain tumor model. In the other study, Daldrup-Link et al.(13) found that ferumoxides labeled CD34-negative hematopoietic stem cells efficiently but CD34-positive stem cells remained unlabeled. Raynal et al. (35) also found that the mechanism of ferumoxides endocytosis was receptor mediated and involved in macrophage scavenger receptors. Thus, phagocytic activity and scavenger receptor expression may be crucial cellular functions necessary for the in vivo uptake of ferumoxides such as those used in the present study.

Transferrin receptor (CD71) is required for iron delivery from transferrin to cells (1, 18, 37). Bulte et al.(10) used CD71 as a vehicle for intracellular monocrystalline iron oxide nanoparticle delivery in oligodendrocyte progenitor cells. Pawelczyk et al. (33) found that Fe-Pro complex in vitro labeling resulted in a transient decrease in CD71 mRNA and protein levels of both HeLa and human mesenchymal stem cells. In contrast, in vitro Fe-Pro complex labeling of primary macrophages resulted in an increase in CD71 mRNA but not in protein levels. Based on our data, the different CD71 staining pattern in iron-labeled cells versus nonlabeled cells may indicate a role for CD71 in iron labeling. This may also be a nonspecific effect of labeling, i.e., excess intracellular iron may downregulate the transferrin receptor.

Serial brain imaging showed that the hypointense MRI signal due to the presence of SPIO-labeled leukocytes and splenocytes became less prominent over time (Fig. 7). This could be due to dilution of iron (through cell division or migration) and/or metabolism of iron oxide particles to undetectable products. The left control hemisphere injected with unlabeled cells only exhibited slightly hypointense regions due to bleeding and/or trauma along the needle track. Therefore, it is unlikely that the observed migration pattern in the right treated hemisphere within the same animal is simply caused by inflammation or trauma. Of interest for others evaluating brain injections, we observed that air bubble injection into the brain could cause similar hypointense signal on MRI as seen with SPIO particles. Unlike SPIO particles or labeled cells, the signal intensity caused by air bubble disappeared 24 h after inoculation.

There are several limitations to this study. First, the dose (20 mg/kg) of ferumoxides in the rat is much higher than the approved dose (0.56 mg/kg) in humans. However, the differences in nanoparticle half-life between rats and humans make it is difficult to extrapolate the rat dose to the human directly. In rats, we safely dose-escalated iron oxide nanoparticles up to 100 mg/kg. High doses are commonly used in animal studies, translating to lower doses in humans. This approach has been successful with USPIO agents for brain tumor imaging (28, 44). Nevertheless, clinical translation of the animal dose will be impossible without a dose escalation study. Second, although both of the component agents in the FE-Pro complex, ferumoxides and protamine sulfate, are FDA approved, neither in vitro labeling nor intravenous injection of the complex is FDA approved for human use. Finally, although protamine sulfate consistently doubled cell labeling in vivo compared with ferumoxides alone, the total magnitude of labeled cells remained low. Additional animal studies are required before this approach could be translated to the clinic.

In summary, we have demonstrated, for the first time, that larger SPIO (ferumoxides) particles but not smaller USPIO particles (ferumoxytol and ferumoxan-10) can iron label mononuclear leukocytes in vivo. From our cell phenotyping and characterization data, CD11b/c and CD45RA (but not CD3) are associated with cellular iron nanoparticle uptake. In terms of practical applications, the advantages of this new in vivo MRI labeling technique will be largely dependent on whether and under what conditions labeled leukocytes may be used to observe trafficking to the CNS from the blood since the ability of labeled cells to circulate and traffic has not yet been demonstrated. This in vivo labeling approach provides an alternative to the in vitro iron labeling technique that has already been tested in humans (14, 47). Further animal studies are in progress to characterize cells trafficking into the CNS from the blood using newly installed 7- and 12-T MR magnets to enhance detection of as few labeled cells as possible with the maximum spatial resolution.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Department of Veterans Affairs and the Biomedical Laboratory Research and Development Service (to E. A. Neuwelt and R. E. Jones), by a Veteran's Administration Merit Review Grant (to E. A. Neuwelt), and by National Institute of Neurological Disorders and Stroke Grants NS-33618, NS-34608, and NS-44687 (to E. A. Neuwelt).


   ACKNOWLEDGMENTS
 
The authors thank Sheila Taylor for the excellent technical support in cell culture and histology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Neuwelt, Dept. of Neurology and Blood-Brain Barrier Program, Oregon Health and Sciences Univ., 3181 Sam Jackson Pkwy., L603, Portland, OR 97239 (e-mail: neuwelte{at}ohsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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