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.

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.

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.