Natalie L. Adolphi

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

IntroductionBreast 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.MethodsTo 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.ResultsWe 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.ConclusionsThese 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.

Targeting and cellular trafficking of magnetic nanoparticles for prostate cancer imaging.

Antibody-conjugated iron oxide nanoparticles offer a specific and sensitive tool to enhance magnetic resonance (MR) images of both local and metastatic cancer. Prostate-specific membrane antigen (PSMA) is predominantly expressed on the neovasculature of solid tumors and on the surface of prostate cells, with enhanced expression following androgen deprivation therapy. Biotinylated anti-PSMA antibody was conjugated to streptavidin-labeled iron oxide nanoparticles and used in MR imaging and confocal laser scanning microscopic imaging studies using LNCaP prostate cancer cells. Labeled iron oxide nanoparticles are internalized by receptor-mediated endocytosis, which involves the formation of clathrin-coated vesicles. Endocytosed particles are not targeted to the Golgi apparatus for recycling but instead accumulate within lysosomes. In T1-weighted MR images, the signal enhancement owing to the magnetic particles was greater for cells with magnetic particles bound to the cell surface than for cells that internalized the particles. However, the location of the particles (surface vs internal) did not significantly alter their effect on T2-weighted images. Our findings indicate that targeting prostate cancer cells using PSMA offers a specific and sensitive technique for enhancing MR images.

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.

Magnetically Responsive Nanoparticles for Drug Delivery Applications Using Low Magnetic Field Strengths

The purpose of this study is to investigate the potential of magnetic nanoparticles for enhancing drug delivery using a low oscillating magnetic field (OMF) strength. We investigated the ability of magnetic nanoparticles to cause disruption of a viscous biopolymer barrier to drug delivery and the potential to induce triggered release of drug conjugated to the surfaces of these particles. Various magnetic nanoparticles were screened for thermal response under a 295-kHz OMF with an amplitude of 3.1 kA/m. Based on thermal activity of particles screened, we selected the nanoparticles that displayed desired characteristics for evaluation in a simplified model of an extracellular barrier to drug delivery, using lambda DNA/HindIII. Results indicate that nanoparticles could be used to induce DNA breakage to enhance local diffusion of drugs, despite low temperatures of heating. Additional studies showed increased diffusion of quantum dots in this model by single-particle tracking methods. Bimane was conjugated to the surface of magnetic nanoparticles. Fluorescence and transmission electron microscope images of the conjugated nanoparticles indicated little change in the overall appearance of the nanoparticles. A release study showed greater drug release using OMF, while maintaining low bulk heating of the samples (T=30degC). This study indicates that lower magnetic field strengths may be successfully utilized for drug delivery applications as a method for drug delivery transport enhancement and drug release switches.

Short data-acquisition times improve projection images of lung tissue.

MR images of laboratory rat lungs that resolve the thin membranes that separate lung lobes are presented. It appears that the capabilities of in vivo small‐animal pulmonary MRI may rival those of in vivo small‐animal X‐ray CT. Free induction decay (FID)‐projection imaging was employed with particular attention to the choice of acquisition time. For a given nominal resolution, one obtains optimal point discrimination when the acquisition time Tacq normalized by the signal decay time constant T  2* is approximately 0.8–0.9, although a better signal‐to‐noise ratio (SNR) is obtained when this quotient is 1.6. Currently available equipment should be able to even exceed the results presented herein. Magn Reson Med 57:1058–1064, 2007.

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.

Preparation and Characterization of Novel Magnetic Nano-in-Microparticles for Site-Specific Pulmonary Drug Delivery

We propose the use of novel inhalable nano-in-microparticles (NIMs) for site-specific pulmonary drug delivery. Conventional lung cancer therapy has failed to achieve therapeutic drug concentrations at tumor sites without causing adverse effects in healthy tissue. To increase targeted drug delivery near lung tumors, we have prepared and characterized a magnetically responsive dry powder vehicle containing doxorubicin. A suspension of lactose, doxorubicin and Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) were spray dried. NIMs were characterized for their size and morphological properties by various techniques: dynamic light scattering (DLS) and laser diffraction (LS) to determine hydrodynamic size of the SPIONs and the NIMs, respectively; next generation cascade impactor (NGI) to determine the aerodynamic diameter and fine particle fraction (FPF); scanning (SEM) and transmission (TEM) electron microscopy to analyze particle surface morphology; electron dispersive X-ray spectroscopy (EDS) to determine iron loading in NIMs; inductively coupled plasma atomic emission spectroscopy (ICP-AES) and superconducting quantum interference device (SQUID) to determine Fe3O4 content in the microparticles; and high performance liquid chromatography (HPLC) to determine doxorubicin loading in the vehicle. NIMs deposition and retention near a magnetic field was performed using a proof-of-concept cylindrical tube to mimic the conducting airway deposition. The hydrodynamic size and zeta potential of SPIONs were 56 nm and -49 mV, respectively. The hydrodynamic and aerodynamic NIM diameters were 1.6 μm and 3.27±1.69 μm, respectively. SEM micrographs reveal spherical particles with rough surface morphology. TEM and focused ion beam-SEM micrographs corroborate the porous nature of NIMs, and surface localization of SPIONs. An in vitro tracheal mimic study demonstrates more than twice the spatial deposition and retention of NIMs, compared to a liquid suspension, in regions under the influence of a strong magnetic gradient. We report the novel formulation of an inhaled and magnetically responsive NIM drug delivery vehicle. This vehicle is capable of being loaded with one or more chemotherapeutic agents, with future translational ability to be targeted to lung tumors using an external magnetic field.

Quantitative mapping of ventilation-perfusion ratios in lungs by 19F MR imaging of T1 of inert fluorinated gases.

A new method is presented for quantitative mapping of ventilation‐to‐perfusion ratios (VA/Q) in the lung: MRI of the 19F longitudinal relaxation time (T1) of an inert fluorinated gas at thermal polarization. The method takes advantage of the dependence of the 19F T1 on the local SF6 partial pressure, which depends on the local value of VA/Q. In contrast to hyperpolarized noble gases, with very long T1s, the T1 of SF6 in mammal lungs is 0.8–1.3 ms. Thus, rapid signal averaging overcomes the low thermal equilibrium polarization. T1 imaging of a phantom consisting of four different SF6/air mixtures with known T1 values validates the modified Look‐Locker T1 imaging sequence. To demonstrate the method in vivo, partial obstruction of the left bronchus was attempted in three rats; 3D free induction decay (FID)‐projection T1 images (2 mm isotropic resolution) revealed obstructed ventilation in two of the animals. In those images, ≈1700 lung voxels contained sufficient SF6 for analysis and T1 was determined in each voxel with a standard error of 8–10%. For comparison, independent VA/Q images of the same animals were obtained using a previously described SF6 MRI technique, and good agreement between the two techniques was obtained. Relative to the previous technique the resolution achieved using the T1 method is lower (for similar VA/Q precision and imaging time); however, the T1 method offers the potential advantages of eliminating the need for image coregistration and allowing patients with impaired lung function to breathe a 70% O2 gas mixture during the entire imaging procedure. Magn Reson Med 59:739–746, 2008.

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.

Iron Oxide Nanocrystals for Magnetic Hyperthermia Applications

Magnetic nanocrystals have been investigated extensively in the past several years for several potential applications, such as information technology, MRI contrast agents, and for drug conjugation and delivery. A specific property of interest in biomedicine is magnetic hyperthermia—an increase in temperature resulting from the thermal energy released by magnetic nanocrystals in an external alternating magnetic field. Iron oxide nanocrystals of various sizes and morphologies were synthesized and tested for specific losses (heating power) using frequencies of 111.1 kHz and 629.2 kHz, and corresponding magnetic field strengths of 9 and 25 mT. Polymorphous nanocrystals as well as spherical nanocrystals and nanowires in paramagnetic to ferromagnetic size range exhibited good heating power. A remarkable 30 °C temperature increase was observed in a nanowire sample at 111 kHz and magnetic field of 25 mT (19.6 kA/m), which is very close to the typical values of 100 kHz and 20 mT used in medical treatments.