Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging.

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs--ferumoxytol, heparin and protamine--in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Phase contrast MRI is an early marker of micrometastatic breast cancer development in the rat brain.

The early growth of micrometastatic breast cancer in the brain often occurs through vessel co-option and is independent of angiogenesis. Remodeling of the existing vasculature is an important step in the evolution of co-opting micrometastases into angiogenesis-dependent solid tumor masses. The purpose of this study was to determine whether phase contrast MRI, an intrinsic source of contrast exquisitely sensitive to the magnetic susceptibility properties of deoxygenated hemoglobin, could detect vascular changes occurring independent of angiogenesis in a rat model of breast cancer metastases to the brain. Twelve nude rats were administered 10(6) MDA-MB-231BRL 'brain-seeking' breast cancer cells through intracardiac injection. Serial, multiparametric MRI of the brain was performed weekly until metastatic disease was detected. The results demonstrated that images of the signal phase (area under the receiver operating characteristic curve, 0.97) were more sensitive than T(2)* gradient echo magnitude images (area under the receiver operating characteristic curve, 0.73) to metastatic brain lesions. The difference between the two techniques was probably the result of the confounding effects of edema on the magnitude of the signal. A region of interest analysis revealed that vascular abnormalities detected with phase contrast MRI preceded tumor permeability measured with contrast-enhanced MRI by 1-2 weeks. Tumor size was correlated with permeability (R(2)= 0.23, p < 0.01), but phase contrast was independent of tumor size (R(2)= 0.03). Histopathologic analysis demonstrated that capillary endothelial cells co-opted by tumor cells were significantly enlarged, but less dense, relative to the normal brain vasculature. Although co-opted vessels were vascular endothelial growth factor-negative, vessels within larger tumor masses were vascular endothelial growth factor-positive. In conclusion, phase contrast MRI is believed to be sensitive to vascular remodeling in co-opting brain tumor metastases independent of sprouting angiogenesis, and may therefore aid in preclinical studies of angiogenic-independent tumors or in the monitoring of continued tumor growth following anti-angiogenic therapy. Published 2011. This article is a US Government work and is in the public domain in the USA.

Differential microstructure and physiology of brain and bone metastases in a rat breast cancer model by diffusion and dynamic contrast enhanced MRI.

Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. In this regard, dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor tumor vascular permeability and cellularity, respectively. In a rat model of metastatic breast cancer, we demonstrate that brain and bone metastases develop with distinct physiological characteristics as measured with MRI. Specifically, brain metastases have limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone had high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlight the influence of the microenvironment on metastatic tumor growth. In light of these results, the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases, respectively, remains a formidable challenge.

Quantification of SPIO nanoparticles in vivo using the finite perturber method.

The susceptibility gradients generated by super-paramagnetic iron oxide (SPIO) nanoparticles make them an ideal contrast agent in magnetic resonance imaging. Traditional quantification methods for SPIO nanoparticle-based contrast agents rely on either mapping T2* values within a region or by modeling the magnetic field inhomogeneities generated by the contrast agent. In this study, a new model-based SPIO quantification method is introduced. The proposed method models magnetic field inhomogeneities by approximating regions containing SPIOs as ensembles of magnetic dipoles, referred to as the finite perturber method. The proposed method was verified using data acquired from a phantom and in vivo mouse models. The phantom consisted of an agar solution with four embedded vials, each vial containing known but different concentrations of SPIO nanoparticles. Gaussian noise was also added to the phantom data to test performance of the proposed method. The in vivo dataset was acquired using five mice, each of which was subcutaneously implanted in the flanks with 1 × 10(5) labeled and 1 × 10(6) unlabeled C6 glioma cells. For the phantom data set, the proposed algorithm was generate accurate estimations of the concentration of SPIOs. For the in vivo dataset, the method was able to give estimations of the concentration within SPIO-labeled tumors that are reasonably close to the known concentration.

Fast T(2) relaxometry with an accelerated multi-echo spin-echo sequence.

A new method has been developed to reduce the number of phase-encoding steps in a multi-echo spin-echo imaging sequence allowing fast T(2) mapping without loss of spatial resolution. In the proposed approach, the k-space data at each echo time were undersampled and a reconstruction algorithm that exploited the temporal correlation of the MR signal in k-space was used to reconstruct alias-free images. A specific application of this algorithm with multiple-receiver acquisition, offering an alternative to existing parallel imaging methods, has also been introduced. The fast T(2) mapping method has been validated in human brain T(2) measurements in a group of nine volunteers with acceleration factors up to 3.4. The results demonstrated that the proposed method exhibited excellent linear correlation with the regular T(2) mapping with full sampling and achieved better image reconstruction and T(2) mapping with respect to SNR and reconstruction artifacts than the selected reference acceleration techniques. The new method has also been applied for quantitative tracking of injected magnetically labeled breast cancer cells in the rat brain with acceleration factors of 1.8 and 3.0. The proposed technique can provide an effective approach for accelerated T(2) quantification, especially for experiments with single-channel coil when parallel imaging is not applicable.

Ultrashort T2* relaxometry for quantitation of highly concentrated superparamagnetic iron oxide (SPIO) nanoparticle labeled cells.

A new method was developed to measure ultrashort T(2)* relaxation in tissues containing a focal area of superparamagnetic iron oxide (SPIO) nanoparticle-labeled cells in which the T(2)* decay is too short to be accurately measured using regular gradient echo T(2)* mapping. The proposed method utilizes the relatively long T(2) relaxation of SPIO-labeled cells and acquires a series of spin echo images with the readout echo shifted to sample the T(2)* decay curve. MRI experiments in phantoms and rats with SPIO-labeled tumors demonstrated that it can detect ultrashort T(2)* down to 1 ms or less. The measured T(2)* values were about 10% higher than those from the ultrashort TE (UTE) technique. The shorter the TE, the less the measurements deviated from the UTE T(2)* mapping. Combined with the regular T(2)* mapping, this technique is expected to provide quantitation of highly concentrated iron-labeled cells from direct cell transplantation.

In vivo MRI using positive-contrast techniques in detection of cells labeled with superparamagnetic iron oxide nanoparticles.

Positive-contrast techniques are being developed to increase the detection of magnetically labeled cells in tissues. We evaluated a post-processing positive-contrast technique, susceptibility-gradient mapping (SGM), and compared this approach with two pulse sequences, a gradient-compensation-based "White Marker" technique and an off-resonance-based approach, inversion recovery on-resonance water suppression (IRON), for the detection of superparamagnetic iron oxide (SPIO) nanoparticle-labeled C6 glioma cells implanted in the flanks of nude rats. The SGM, White Marker and IRON positive-contrast images were acquired when the labeled C6 glioma tumors were approximately 5 mm (small), approximately 10 mm (medium) and approximately 20 mm (large) in diameter along the largest dimension to evaluate their sensitivity to the dilution of the SPIO nanoparticles as the tumor cells proliferated. In vivo MRI demonstrated that all three positive-contrast techniques can produce hyperintensities in areas around the labeled flank tumors against a dark background. The number of positive voxels detected around small and medium tumors was significantly greater with the SGM technique than with the White Marker and IRON techniques. For large tumors, the SGM resulted in a similar number of positive voxels to the White Marker technique, and the IRON approach failed to generate positive-contrast images with a 200 Hz suppression band. This study also reveals that hemorrhage appears as hyperintensities on positive-contrast images and may interfere with the detection of SPIO-labeled cells.

Color transformation and fluorescence of Prussian blue-positive cells: implications for histologic verification of cells labeled with superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide (SPIO) nanoparticles, either modified or in combination with other macromolecules, are being used for magnetic labeling of stem cells and other cells to monitor cell trafficking by magnetic resonance imaging (MRI) in experimental models. The correlation of histology to MRI depends on the ability to detect SPIO-labeled cells using Prussian blue (PB) stain and fluorescent tags to cell surface markers. Exposure of PB-positive sections to ultraviolet light at a wavelength of 365 nm commonly used fluorescence microscopy can result in color transformation of PB-positive material from blue to brown. Although the PB color transformation is primarily an artifact that may occur during fluorescence microscopy, the transformation can be manipulated using imaging process software for the detection of low levels of iron labeled cells in tissues samples.

Effect of human stem cells labeled with ferumoxides-poly-L-lysine on hematologic and biochemical measurements in rats.

PURPOSE: To determine whether ferumoxides-poly-l-lysine (PLL) complex-labeled mesenchymal stem cells (MSCs) or ferumoxides-PLL complex alone alters hematologic, blood chemistry, renal function, and/or liver function measurements after being intravenously infused into rats. MATERIALS AND METHODS: Twenty-five rats (group 1) received intravenous injections of labeled MSCs, and 25 additional rats (group 2) received intravenous injections of ferumoxides-PLL complex only. Complete blood counts, liver and renal function test results, and serum electrolyte and iron concentrations were measured for 42 days after the injections and compared with those measured in five control rats (group 3). To determine the duration of labeled MSCs in the circulation, venous blood was serially drawn from five additional rats (group 4) that were injected with labeled MSCs. Analyses of variance (ANOVA) followed by Fisher protected least significant difference post hoc tests were used to statistically analyze results. P < .05 was considered to indicate significance in all analyses. RESULTS: Administration of neither labeled MSCs nor ferumoxides-PLL complex had a significant effect on hematologic or blood chemistry indicators of organ function. Of the parameters measured, only hemoglobin concentration and mean corpuscular volume (MCV) in the rats injected with labeled MSCs, as well as MCV and hemoglobin, alkaline phosphatase, aspartate aminotransferase, and direct bilirubin concentrations in the rats injected with ferumoxides-PLL complex, varied significantly during the 42-day postinjection period (P < .05, ANOVA). No other measurements, including serum electrolyte and iron concentrations, changed significantly during the test period (P > .05). Furthermore, injected labeled MSCs had cleared from the peripheral circulation by 15 minutes after injection. CONCLUSION: Results indicate that infusing cells that are magnetically labeled with ferumoxides-PLL complex into rats does not alter biochemical or hematologic measures of organ function in a clinically relevant or preclusive manner.

Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents.

PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents. MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated. RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells. CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.