Development of advanced signal processing and source imaging methods for superparamagnetic relaxometry.

Superparamagnetic relaxometry (SPMR) is a highly sensitive technique for the in vivo detection of tumor cells and may improve early stage detection of cancers. SPMR employs superparamagnetic iron oxide nanoparticles (SPION). After a brief magnetizing pulse is used to align the SPION, SPMR measures the time decay of SPION using super-conducting quantum interference device (SQUID) sensors. Substantial research has been carried out in developing the SQUID hardware and in improving the properties of the SPION. However, little research has been done in the pre-processing of sensor signals and post-processing source modeling in SPMR. In the present study, we illustrate new pre-processing tools that were developed to: (1) remove trials contaminated with artifacts, (2) evaluate and ensure that a single decay process associated with bounded SPION exists in the data, (3) automatically detect and correct flux jumps, and (4) accurately fit the sensor signals with different decay models. Furthermore, we developed an automated approach based on multi-start dipole imaging technique to obtain the locations and magnitudes of multiple magnetic sources, without initial guesses from the users. A regularization process was implemented to solve the ambiguity issue related to the SPMR source variables. A procedure based on reduced chi-square cost-function was introduced to objectively obtain the adequate number of dipoles that describe the data. The new pre-processing tools and multi-start source imaging approach have been successfully evaluated using phantom data. In conclusion, these tools and multi-start source modeling approach substantially enhance the accuracy and sensitivity in detecting and localizing sources from the SPMR signals. Furthermore, multi-start approach with regularization provided robust and accurate solutions for a poor SNR condition similar to the SPMR detection sensitivity in the order of 1000 cells. We believe such algorithms will help establishing the industrial standards for SPMR when applying the technique in pre-clinical and clinical settings.

Magnetic relaxometry as applied to sensitive cancer detection and localization.

BACKGROUND: Here we describe superparamagnetic relaxometry (SPMR), a technology that utilizes highly sensitive magnetic sensors and superparamagnetic nanoparticles for cancer detection. Using SPMR, we sensitively and specifically detect nanoparticles conjugated to biomarkers for various types of cancer. SPMR offers high contrast in vivo, as there is no superparamagnetic background, and bones and tissue are transparent to the magnetic fields. METHODS: In SPMR measurements, a brief magnetizing pulse is used to align superparamagnetic nanoparticles of a discrete size. Following the pulse, an array of superconducting quantum interference detectors (SQUID) sensors detect the decaying magnetization field. NP size is chosen so that, when bound, the induced field decays in seconds. They are functionalized with specific biomarkers and incubated with cancer cells in vitro to determine specificity and cell binding. For in vivo experiments, functionalized NPs are injected into mice with xenograft tumors, and field maps are generated to localize tumor sites. RESULTS: Superparamagnetic NPs developed here have small size dispersion. Cell incubation studies measure specificity for different cell lines and antibodies with very high contrast. In vivo animal measurements verify SPMR localization of tumors. Our results indicate that SPMR possesses sensitivity more than 2 orders of magnitude better than previously reported.

Modeling the efficiency of a magnetic needle for collecting magnetic cells.

As new magnetic nanoparticle-based technologies are developed and new target cells are identified, there is a critical need to understand the features important for magnetic isolation of specific cells in fluids, an increasingly important tool in disease research and diagnosis. To investigate magnetic cell collection, cell-sized spherical microparticles, coated with superparamagnetic nanoparticles, were suspended in (1) glycerine-water solutions, chosen to approximate the range of viscosities of bone marrow, and (2) water in which 3, 5, 10 and 100% of the total suspended microspheres are coated with magnetic nanoparticles, to model collection of rare magnetic nanoparticle-coated cells from a mixture of cells in a fluid. The magnetic microspheres were collected on a magnetic needle, and we demonstrate that the collection efficiency versus time can be modeled using a simple, heuristically-derived function, with three physically-significant parameters. The function enables experimentally-obtained collection efficiencies to be scaled to extract the effective drag of the suspending medium. The results of this analysis demonstrate that the effective drag scales linearly with fluid viscosity, as expected. Surprisingly, increasing the number of non-magnetic microspheres in the suspending fluid results increases the collection of magnetic microspheres, corresponding to a decrease in the effective drag of the medium.

Development of antibody-tagged nanoparticles for detection of transplant rejection using biomagnetic sensors.

Organ transplantation is a life-saving procedure and the preferred method of treatment for a growing number of disease states. The advent of new immunosuppressants and improved care has led to great advances in both patient and graft survival. However, acute T-cell-mediated graft rejection occurs in a significant quantity of recipients and remains a life-threatening condition. Acute rejection is associated with decrease in long-term graft survival, demonstrating a need to carefully monitor transplant patients. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies or relatively nonspecific clinical features. A noninvasive way is needed to detect, localize, and monitor transplant rejection. Capitalizing on advances in targeted contrast agents and magnetic-based detection technology, we developed anti-CD3 antibody-tagged nanoparticles. T cells were found to bind preferentially to antibody-tagged nanoparticles, as identified through light microscopy, transmission electron microscopy, and confocal microscopy. Using mouse skin graft models, we were also able to demonstrate in vivo vascular delivery of T-cell targeted nanoparticles. We conclude that targeting lymphocytes with magnetic nanoparticles is conducive to developing a novel, noninvasive strategy for identifying transplant rejection.

Magnetic Relaxometry with an Atomic Magnetometer and SQUID Sensors on Targeted Cancer Cells.

Magnetic relaxometry methods have been shown to be very sensitive in detecting cancer cells and other targeted diseases. Superconducting Quantum Interference Device (SQUID) sensors are one of the primary sensor systems used in this methodology because of their high sensitivity with demonstrated capabilities of detecting fewer than 100,000 magnetically-labeled cancer cells. The emerging technology of atomic magnetometers (AM) represents a new detection method for magnetic relaxometry with high sensitivity and without the requirement for cryogens. We report here on a study of magnetic relaxometry using both AM and SQUID sensors to detect cancer cells that are coated with superparamagnetic nanoparticles through antibody targeting. The AM studies conform closely to SQUID sensor results in the measurement of the magnetic decay characteristics following a magnetization pulse. The AM and SQUID sensor data are well described theoretically for superparamagnetic particles bound to cells and the results can be used to determine the number of cells in a cell culture or tumor. The observed fields and magnetic moments of cancer cells are linear with the number of cells over a very large range. The AM sensor demonstrates very high sensitivity for detecting magnetically labeled cells does not require cryogenic cooling and is relatively inexpensive.

Imaging of Her2-targeted magnetic nanoparticles for breast cancer detection: comparison of SQUID-detected magnetic relaxometry and MRI.

Both magnetic relaxometry and magnetic resonance imaging (MRI) can be used to detect and locate targeted magnetic nanoparticles, noninvasively and without ionizing radiation. Magnetic relaxometry offers advantages in terms of its specificity (only nanoparticles are detected) and the linear dependence of the relaxometry signal on the number of nanoparticles present. In this study, detection of single-core iron oxide nanoparticles by superconducting quantum interference device (SQUID)-detected magnetic relaxometry and standard 4.7 T MRI are compared. The nanoparticles were conjugated to a Her2 monoclonal antibody and targeted to Her2-expressing MCF7/Her2-18 (breast cancer cells); binding of the nanoparticles to the cells was assessed by magnetic relaxometry and iron assay. The same nanoparticle-labeled cells, serially diluted, were used to assess the detection limits and MR relaxivities. The detection limit of magnetic relaxometry was 125 000 nanoparticle-labeled cells at 3 cm from the SQUID sensors. T(2)-weighted MRI yielded a detection limit of 15 600 cells in a 150 µl volume, with r(1) = 1.1 mm(-1) s(-1) and r(2) = 166 mm(-1) s(-1). Her2-targeted nanoparticles were directly injected into xenograft MCF7/Her2-18 tumors in nude mice, and magnetic relaxometry imaging and 4.7 T MRI were performed, enabling direct comparison of the two techniques. Co-registration of relaxometry images and MRI of mice resulted in good agreement. A method for obtaining accurate quantification of microgram quantities of iron in the tumors and liver by relaxometry was also demonstrated. These results demonstrate the potential of SQUID-detected magnetic relaxometry imaging for the specific detection of breast cancer and the monitoring of magnetic nanoparticle-based therapies.

Detection of breast cancer cells using targeted magnetic nanoparticles and ultra-sensitive magnetic field sensors.

INTRODUCTION: Breast cancer detection using mammography has improved clinical outcomes for many women, because mammography can detect very small (5 mm) tumors early in the course of the disease. However, mammography fails to detect 10 - 25% of tumors, and the results do not distinguish benign and malignant tumors. Reducing the false positive rate, even by a modest 10%, while improving the sensitivity, will lead to improved screening, and is a desirable and attainable goal. The emerging application of magnetic relaxometry, in particular using superconducting quantum interference device (SQUID) sensors, is fast and potentially more specific than mammography because it is designed to detect tumor-targeted iron oxide magnetic nanoparticles. Furthermore, magnetic relaxometry is theoretically more specific than MRI detection, because only target-bound nanoparticles are detected. Our group is developing antibody-conjugated magnetic nanoparticles targeted to breast cancer cells that can be detected using magnetic relaxometry. METHODS: To accomplish this, we identified a series of breast cancer cell lines expressing varying levels of the plasma membrane-expressed human epidermal growth factor-like receptor 2 (Her2) by flow cytometry. Anti-Her2 antibody was then conjugated to superparamagnetic iron oxide nanoparticles using the carbodiimide method. Labeled nanoparticles were incubated with breast cancer cell lines and visualized by confocal microscopy, Prussian blue histochemistry, and magnetic relaxometry. RESULTS: We demonstrated a time- and antigen concentration-dependent increase in the number of antibody-conjugated nanoparticles bound to cells. Next, anti Her2-conjugated nanoparticles injected into highly Her2-expressing tumor xenograft explants yielded a significantly higher SQUID relaxometry signal relative to unconjugated nanoparticles. Finally, labeled cells introduced into breast phantoms were measured by magnetic relaxometry, and as few as 1 million labeled cells were detected at a distance of 4.5 cm using our early prototype system. CONCLUSIONS: These results suggest that the antibody-conjugated magnetic nanoparticles are promising reagents to apply to in vivo breast tumor cell detection, and that SQUID-detected magnetic relaxometry is a viable, rapid, and highly sensitive method for in vitro nanoparticle development and eventual in vivo tumor detection.

Magnetic Properties of Nanoparticles Useful for SQUID Relaxometry in Biomedical Applications.

We use dynamic susceptometry measurements to extract semiempirical temperature-dependent, 255 to 400 K, magnetic parameters that determine the behavior of single-core nanoparticles useful for SQUID relaxometry in biomedical applications. Volume susceptibility measurements were made in 5K degree steps at nine frequencies in the 0.1 - 1000 Hz range, with a 0.2 mT amplitude probe field. The saturation magnetization (M(s)) and anisotropy energy density (K) derived from the fitting of theoretical susceptibility to the measurements both increase with decreasing temperature; good agreement between the parameter values derived separately from the real and imaginary components is obtained. Characterization of the Néel relaxation time indicates that the conventional prefactor, 0.1 ns, is an upper limit, strongly correlated with the anisotropy energy density. This prefactor decreases substantially for lower temperatures, as K increases. We find, using the values of the parameters determined from the real part of the susceptibility measurements at 300 K, that SQUID relaxometry measurements of relaxation and excitation curves on the same sample are well described.

Characterization of single-core magnetite nanoparticles for magnetic imaging by SQUID relaxometry.

Optimizing the sensitivity of SQUID (superconducting quantum interference device) relaxometry for detecting cell-targeted magnetic nanoparticles for in vivo diagnostics requires nanoparticles with a narrow particle size distribution to ensure that the Néel relaxation times fall within the measurement timescale (50 ms-2 s, in this work). To determine the optimum particle size, single-core magnetite nanoparticles (with nominal average diameters 20, 25, 30 and 35 nm) were characterized by SQUID relaxometry, transmission electron microscopy, SQUID susceptometry, dynamic light scattering and zeta potential analysis. The SQUID relaxometry signal (detected magnetic moment/kg) from both the 25 nm and 30 nm particles was an improvement over previously studied multi-core particles. However, the detected moments were an order of magnitude lower than predicted based on a simple model that takes into account the measured size distributions (but neglects dipolar interactions and polydispersity of the anisotropy energy density), indicating that improved control of several different nanoparticle properties (size, shape and coating thickness) will be required to achieve the highest detection sensitivity. Antibody conjugation and cell incubation experiments show that single-core particles enable a higher detected moment per cell, but also demonstrate the need for improved surface treatments to mitigate aggregation and improve specificity.

Enhanced leukemia cell detection using a novel magnetic needle and nanoparticles.

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34-conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care.

Characterization of magnetite nanoparticles for SQUID-relaxometry and magnetic needle biopsy.

Magnetite nanoparticles (Chemicell SiMAG-TCL) were characterized by SQUID-relaxometry, susceptometry, and TEM. The magnetization detected by SQUID-relaxometry was 0.33% of that detected by susceptometry, indicating that the sensitivity of SQUID-relaxometry could be significantly increased through improved control of nanoparticle size. The relaxometry data were analyzed by the moment superposition model (MSM) to determine the distribution of nanoparticle moments. Analysis of the binding of CD34-conjugated nanoparticles to U937 leukemia cells revealed 60,000 nanoparticles per cell, which were collected from whole blood using a prototype magnetic biopsy needle, with a capture efficiency of >65% from a 750 µl sample volume in 1 minute.

Use of a SQUID array to detect T-cells with magnetic nanoparticles in determining transplant rejection.

Acute rejection in organ transplant is signaled by the proliferation of T-cells that target and kill the donor cells requiring painful biopsies to detect rejection onset. An alternative non-invasive technique is proposed using a multi-channel superconducting quantum interference device (SQUID) magnetometer to detect T-cell lymphocytes in the transplanted organ labeled with magnetic nanoparticles conjugated to antibodies specifically attached to lymphocytic ligand receptors. After a magnetic field pulse, the T-cells produce a decaying magnetic signal with a characteristic time of the order of a second. The extreme sensitivity of this technique, 10(5) cells, can provide early warning of impending transplant rejection and monitor immune-suppressive chemotherapy.

Magnetic needles and superparamagnetic cells.

Superparamagnetic nanoparticles can be attached in great numbers to pathogenic cells using specific antibodies so that the magnetically-labeled cells themselves become superparamagnets. The cells can then be manipulated and drawn out of biological fluids, as in a biopsy, very selectively using a magnetic needle. We examine the origins and uncertainties in the forces exerted on magnetic nanoparticles by static magnetic fields, leading to a model for trajectories and collection times of dilute superparamagnetic cells in biological fluids. We discuss the design and application of such magnetic needles and the theory of collection times. We compare the mathematical model to measurements in a variety of media including blood. For more information on this article, see medicalphysicsweb.org.