Sarah Fredriksson

99mTc-Labeled Superparamagnetic Iron Oxide Nanoparticles for Multimodality SPECT/MRI of Sentinel Lymph Nodes

The purpose of this study was to develop multimodality SPECT/MRI contrast agents for sentinel lymph node (SLN) mapping in vivo. Methods: Nanoparticles with a solid iron oxide core and a polyethylene glycol coating were labeled with 99mTc. The labeling efficiency was determined with instant thin-layer chromatography and magnetic separation. The stability of the radiolabeled superparamagnetic iron oxide nanoparticles (SPIONs) was verified in both sterile water and human serum at room temperature 6 and 24 h after labeling. Five Wistar rats were injected subcutaneously in the right hind paw with 99mTc-SPIONs (25–50 MBq, ∼0.2 mg of Fe) and sacrificed 4 h after injection. Two animals were imaged with SPECT/MRI. All 5 rats were dissected; the lymph nodes, liver, kidneys, spleen, and hind paw containing the injection site were removed and weighed; and activity in the samples was measured. The microdistribution within the lymph nodes was studied with digital autoradiography. Results: The efficiency of labeling of the SPIONs was 99% 6 h after labeling in both water and human serum. The labeling yield was 98% in water and 97% in human serum 24 h after labeling. The SLN could be identified in vivo with SPECT/MRI. The accumulation of 99mTc-SPIONs (as the percentage injected dose/g [%ID/g]) in the SLN was 100 %ID/g, whereas in the liver and spleen it was less than 2 %ID/g. Digital autoradiography images revealed a nonhomogeneous distribution of 99mTc-SPIONs within the lymph nodes; nanoparticles were found in the cortical, subcapsular, and medullary sinuses. Conclusion: This study revealed the feasibility of labeling SPIONs with 99mTc. The accumulation of 99mTc-SPIONs in lymph nodes after subcutaneous injection in animals, verified by SPECT/MRI, is encouraging for applications in breast cancer and malignant melanoma.

EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.

Frequency- and phase-sensitive magnetomotive ultrasound imaging of superparamagnetic iron oxide nanoparticles

It has recently been demonstrated that superparamagnetic iron oxide nanoparticles can be used as magnetomotive ultrasound contrast agents. A time-varying external magnetic field acts to move the particles and, thus, the nanoparticle-laden tissue. However, the difficulty of distinguishing this magnetomotive motion from undesired movement induced in regions without nanoparticles or other motion artifacts has not been well reported. Using a high-frequency linear-array system, we found that displacements outside nanoparticle-laden regions can be similar in magnitude to those in regions containing nanoparticles. We also found that the displacement outside the nanoparticle regions had a phase shift of approximately π radians relative to that in the nanoparticle regions. To suppress signals arising from undesirable movements, we developed an algorithm based on quadrature detection and phase gating at the precise frequency of nanoparticle displacement. Thus, clutter at other frequencies can be filtered out, and the processed signal can be color-coded and superimposed on the B-mode image. The median signal-to-clutter ratio improvement using the proposed algorithm was 36 dB compared with simply summing the movement energy at all frequencies. This clutter rejection is a crucial step to move magnetomotive ultrasound imaging of nanoparticles toward in vivo investigations.

Multimodal Detection of Iron Oxide Nanoparticles in Rat Lymph Nodes Using Magnetomotive Ultrasound Imaging and Magnetic Resonance Imaging

Detection and removal of sentinel lymph nodes (SLN) is important in the diagnosis and treatment of cancer. The SLN is the first regional lymph node draining the primary tumor, and if the cancer has spread, it is most likely to find metastases in the SLN. In this study, we have for the first time been able to image the very same contrast agent, superparamagnetic iron oxide nanoparticles (SPIO-NPs), in rat SLNs by using both our frequency- and phase-gated magnetomotive ultrasound (MMUS) algorithm and conventional magnetic resonance imaging (MRI); MMUS post mortem, MRI in vivo. For both higher NP-concentration and smaller NPs, we found that the MMUS data showed a larger magnetomotive displacement (1.56 ± 0.43 and 1.94 ± 0.54 times larger, respectively) and that the MR-images were affected to a higher degree. The MMUS displacement also increased with lower excitation frequency (1.95 ± 0.64 times larger for 5 Hz compared with 15 Hz) and higher excitation voltage (2.95 ± 1.44 times larger for 30 V compared with 10 V). The results show that MMUS has potential to be used as bedside guidance during SLN surgery, imaging the same particles that were used in prior staging with other imaging techniques.

Optimizing retention of multimodal imaging nanostructures in sentinel lymph nodes by nanoscale size tailoring.

UNLABELLED This study investigates the retention of different sized ultra-small superparamagnetic iron oxide nanoparticles (USPIOs) in lymph nodes of healthy rats, after subcutaneous injection. Three distinct sizes (15, 27 and 58 nm) of USPIOs were synthesized by only varying the thickness of the polymer coating surrounding the 10 nm cores. Particles were injected on the dorsal side of the hind paw of rats and the uptake in the popliteal, inguinal and iliac lymph nodes was monitored. The data reveal that the 15 nm particle accumulates more rapidly and to a higher amount in the first lymph node than the two larger particles. A clear contrast between the first and second lymph nodes could be detected indicating that even the rather small difference in particle size (15-58 nm) tested has significant effects on the retention of USPIOs in the lymph nodes. FROM THE CLINICAL EDITOR From the Clinical Editor: In this study, the size-dependence of USPIO particles is studied from the standpoint of their accumulation characteristics in lymph nodes. The authors conclude that the smaller particles accumulated faster and at a higher concentration than the two larger sizes studied.

Combined Magnetomotive ultrasound, PET/CT, and MR imaging of 68 Ga-labelled superparamagnetic iron oxide nanoparticles in rat sentinel lymph nodes in vivo

Current methods for intra-surgical guidance to localize metastases at cancer surgery are based on radioactive tracers that cause logistical challenges. We propose the use of a novel ultrasound-based method, magnetomotive ultrasound (MMUS) imaging that employ a nanoparticle-based contrast agent that also may be used for pre-operative PET/MRI imaging. Since MMUS is radiation free, this eliminates the dependence between pre- and intra-operative imaging and the radiation exposure for the surgical staff. This study investigates a hypothetical clinical scenario of pre-operative PET imaging, combined with intra-operative MMUS imaging, implemented in a sentinel lymph node (SLN) rat model. At one-hour post injection of 68Ga-labelled magnetic nanoparticles, six animals were imaged with combined PET/CT. After two or four days, the same animals were imaged with MMUS. In addition, ex-vivo MRI was used to evaluate the amount of nanoparticles in each single SLN. All SLNs were detectable by PET. Four out of six SLNs could be detected with MMUS, and for these MMUS and MRI measurements were in close agreement. The MRI measurements revealed that the two SLNs undetectable with MMUS contained the lowest nanoparticle concentrations. This study shows that MMUS can complement standard pre-operative imaging by providing bedside real-time images with high spatial resolution.

Induced tissue displacement in magnetomotive ultrasound imaging - simulations and experiments

Magnetomotive ultrasound imaging is an emerging technique where superparamagnetic iron oxide nanoparticles can be used as an ultrasound contrast agent. A time-varying external magnetic field acts to move the particles lodged in tissue, and ultrasound is used to detect the resulting tissue movement. In phantom studies we have observed opposite phase motion next to regions containing nanoparticles. We hypothesize that this motion is caused by mechanical coupling from regions where nanoparticles are located. The present study compares experimental data to a numerical simulation with identical geometry as the experimental set-up. The magnetic force acting on particles was modeled as emanating from a coil with a cone shaped iron core, and applied as a body load in nanoparticle-laden regions. The simulation showed opposed motion in-between nanoparticle-laden phantom inserts, in a manner similar to the experimental situation. There is a slight mismatch in the extent of vertical movement, which we interpret as a result of the modeled slip condition tangentially to the surface, which in reality presumably is a combination of slip and stick due to friction.

Phase-locked magnetomotive ultrasound imaging of superparamagnetic iron-oxide nanoparticles

It has recently been shown that superparamagnetic iron oxide nanoparticles (SPIO-NP) can be used as ultrasound contrast agents, by moving the particles using a time-varying external magnetic field and thereby moving nanoparticle laden tissue. However, the difficulty to distinguish magneto-motive motion from indirect motion induced outside areas containing nanoparticles, or other motion artifacts, which restrict the use in vivo, is not well reported. Using a high-frequency linear array system we found that displacements outside nanoparticle-laden areas could be in the same range as the displacement in areas containing nanoparticles. We also found in our setup that the movement in areas between the nanoparticles areas were approximately π radians out of phase relative the nanoparticles areas. To suppress undesirable signals we developed an algorithm based on frequency-tracking and phase-locking, where the energy of the processed signal is color-coded and overlaid on the B-mode image. The signal-to-clutter ratio showed a 14.5 dB improvement between the image color-coded with the total power and the image coded with regard to phase and frequency. The capability to distinguish areas containing SPIO-NP from those that do not is a crucial step for detection of nanoparticles in vivo.

Normalization of Magnetic Field Effects: Towards Quantitative Magnetomotive Ultrasound Imaging

In magnetomotive ultrasound (MMUS) imaging superparamagnetic iron oxide nanoparticles (NP) are used as contrast agents and a time-varying external magnetic field acts to move the particles and thereby the nanoparticle-laden tissue. Recently we proposed a frequency and phase sensitive algorithm to reduce motion artifacts. However, the method is not quantitative as the particle movement induced is dependent not only on the field strength, but also on the field gradient, plus material parameters. Here we assess the measured nanoparticle movement across the image plane in comparison with simulations of the magnetic force, to evaluate the potential for image normalization of magnetic field effects. We found that the movement decreased with distance to the iron core tip, from which the magnetic field was extending, and approaches zero at the transducer face. This finding did not coincide with the simulation and may make it difficult to enable quantification. The coefficient of variation between measurements on the homogeneous phantom was typically in the order of 15% for all frequencies, indicating the expected accuracy for quantitative measurements.

In vivo magnetomotive ultrasound imaging of rat lymph nodes - a pilot study

The drive to gain a better understanding of how diseases arise and how to provide ever-earlier detection are some of the key factors for the development of molecular imaging. Compared to other imaging modalities ultrasound has not received the same attention for molecular imaging mainly due to its limited contrast resolution, together with contrast agents confined to the intravascular space. To overcome these issues, new nano-sized contrast agents and new ultrasound imaging techniques e.g. photo acoustic imaging, have been developed. Another such imaging technique under development is magnetomotive ultrasound imaging (MMUS). We have previously developed a frequency and phase tracking algorithm which is able to detect superparamagnetic iron oxide nanoparticles (SPIO NPs) using MMUS, where our suggested first clinical application is to detect sentinel lymph nodes (SLNs) in breast cancer surgery. Recently we have shown detection of SPIO laden rat SLNs in situ. Here we present the feasibility of in vivo detection of SLNs in rats. The algorithm clearly pinpoints the NP laden SLN, even in presence of significant artefactual tissue movement. The magnetomotive displacement increased when a higher voltage was applied on the coil creating the magnetic field (e.g. 56.6% increasing the voltage from 20V to 50V). An uneven concentration distribution of NPs in the SLN was found. The maximum magnetomotive displacement difference between two different cross sections in one SLN was 9.76 times. The study also showed that for a higher concentration of NPs a lower magnetic coil excitation voltage could be used in order to create a magnetomotive displacement of a certain magnitude. The result from this in vivo study shows that the method has potential for future clinical use.