Dale L. Huber

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.

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.

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.

Interactions of endoglucanases with amorphous cellulose films resolved by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.

Poly(N-isopropylacrylamide) Surfactant-Functionalized Responsive Silver Nanoparticles and Superlattices

Metal nanoparticles exhibit unique optical characteristics in visible spectra produced by local surface plasmon resonance (SPR) for a wide range of optical and electronic applications. We report the synthesis of poly(N-isopropylacrylamide) surfactant (PNIPAM-C18)-functionalized metal nanoparticles and ordered superlattice arrays through an interfacial self-assembly process. The method is simple and reliable without using complex chemistry. The PNIPAM-C18-functionalized metal nanoparticles and ordered superlattices exhibit responsive behavior modulated by external temperature and relative humidity (RH). In situ grazing-incidence small-angle X-ray scattering studies confirmed that the superlattice structure of PNIPAM-C18 surfactant-functionalized nanoparticle arrays shrink and spring back reversibly based on external thermal and RH conditions, which allow flexible manipulation of interparticle spacing for tunable SPR. PNIPAM-C18 surfactants play a key role in accomplishing this responsive property. The ease of fabrication of the responsive nanostructure facilitates investigation of nanoparticle coupling that depends on interparticle separation for potential applications in chemical and biological sensors as well as energy storage devices.

Reversible Control of Electrochemical Properties Using Thermally‐Responsive Polymer Electrolytes

A thermally responsive copolymer is designed to modulate the properties of an electrolyte solution. The copolymer is prepared using pNIPAM, which governs the thermal properties, and acrylic acid, which provides the electrolyte ions. As the polymer undergoes a thermally activated phase transition, the local environment around the acid groups is reversibly switched, decreasing ion concentration and conductivity. The responsive electrolyte is used to control the activity of redox electrodes with temperature.

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.

Self-consistent field simulations of self- and directed-assembly in a mixed polymer brush

While self-assembling block copolymer thin films have attracted attention as a promising high resolution lithographic tool, the self assembly of mixed polymer brushes for lithography is relatively unexplored. Here we study the directed self-assembly of a mixed polymer brush using self-consistent field theory (SCFT) simulations. Using the model equations and numerical methods introduced and verified in our previous study, the bulk phase behavior of a mixed melt brush is studied in depth through full three dimensional calculations. We assume that the mixed A/B polymer chains, which are of the same length, are exposed to a neutral top surface and are uniformly grafted at a high density. We identify phase-separated morphologies and calculate a phase diagram for the mixed brush under melt conditions as a function of the segregation force and composition. The observed lateral microphase separation is similar to that in block copolymer thin films, but the phase separation occurs at a smaller segregation force and the transition between cylindrical and spherical morphologies are quite different than the first-order phase transition in block copolymers. We demonstrate that lateral confinement can induce long-range, in-plane order in mixed brushes and suggest promising directed self-assembly methods for the application of self-assembled mixed polymer brushes in next-generation information storage and electronic devices.

Structural and magnetic characterization of superparamagnetic iron platinum nanoparticle contrast agents for magnetic resonance imaging.

We report the synthesis, from simple salts, and the physical characterization of superparamagnetic iron platinum nanoparticles (SIPPs) suitable for use as contrast agents in magnetic resonance imaging. The properties of these particles were determined by means of transmission electron microscopy (TEM), thermogravimetric analysis (TGA), inductively coupled plasma-optical emission spectroscopy (ICP-OES), superconducting quantum interference device (SQUID) magnetometry, and nuclear magnetic resonance (NMR) relaxivity at 4.7 Tesla. TEM showed that the diameters of the particles ranged from 9.3 nm to 10 nm, depending on the mole ratio of iron to platinum precursors, and on the concentration of Octadecylamine (ODA) used in their preparation. The iron to platinum stoichiometry determined by ICP-OES varied from 1.4:1 to 3.7:1 and was similarly dependant on the initial mole ratios of iron and platinum salts, as well as on the concentration of ODA in the reaction. SQUID magnetometry showed that the SIPPs were superparamagnetic and had magnetic moments that increased with increasing iron content from 62 to 72 A•m(2)/kg Fe. The measured relaxivities of the SIPPs at 4.7 Tesla were higher than commercially available superparamagnetic iron oxide nanoparticles (SPIONs), suggesting that these particles may be superior contrast agents in T(2)-weighted magnetic resonance imaging (MRI).

Highly stable multi-anchored magnetic nanoparticles for optical imaging within biofilms.

Magnetic nanoparticles are the next tool in medical diagnoses and treatment in many different biomedical applications, including magnetic hyperthermia as alternative treatment for cancer and bacterial infections, as well as the disruption of biofilms. The colloidal stability of the magnetic nanoparticles in a biological environment is crucial for efficient delivery. A surface that can be easily modifiable can also improve the delivery and imaging properties of the magnetic nanoparticle by adding targeting and imaging moieties, providing a platform for additional modification. The strategy presented in this work includes multiple nitroDOPA anchors for robust binding to the surface tied to the same polymer backbone as multiple poly(ethylene oxide) chains for steric stability. This approach provides biocompatibility and enhanced stability in fetal bovine serum (FBS) and phosphate buffer saline (PBS). As a proof of concept, these polymer-particles complexes were then modified with a near infrared dye and utilized in characterizing the integration of magnetic nanoparticles in biofilms. The work presented in this manuscript describes the synthesis and characterization of a nontoxic platform for the labeling of near IR-dyes for bioimaging.