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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.

labeled nanometer sized colloids are injected sub-dermally or peri-tumourally, and then imaged using a gamma camera/SPECT (before surgery) and a gamma probe (during surgery). A blue dye injected prior to surgery may serve to visualize the SLN intra-operatively. When the SLN is found, it is removed by invasive surgery and cancer infiltration is examined using histology. The lack of precise anatomic information in the scintigraphic images and the non-specificity of the tracer, however, often limit the preoperative planning and the identification of the SLN. Multimodal approaches in the clinic, such as the next generation hybrid systems based on combined MR/PETsystems, are expected to address several of these issues. In the pre-clinical setting, an even faster growing segment for medical imaging‐multimodal systems including optical methods is concurrently emerging. Interestingly, there exist still no commercially available clinical contrast agent that can be used for even two modalities, and research has shown functionality for only a couple imaging modalities at a time. Our aim is to develop novel multimodality contrast agents and methodology for such systems to meet new needs for quantitative and high-resolution imaging. In this perspective, superparamagnetic iron oxide nanoparticles (SPIO-NPs) are emerging as a highly attractive approach. SPIO-NPs can be observed by MRI as signal voids in the MR image and SPIO-NPs have been approved as MRI contrast agents for more than a decade with applications including lymph node detection. We propose the use of SPIO-NPs as a multimodal contrast agent by attaching radionuclides and fluorophores to the surface of the nanoparticles, for, respectively, SPECT, PET, and optical imaging methods. Moreover, the particles also inherently functions as contrast media for a new imaging technique called magnetomotive ultrasound imaging. Thus, the very same particles can be used as a contrast agent for up to five modalities, depending on the type of surface modification. We expect the following improvements for the work-flow of cancer patients undergoing lymph node surgery: • One single injection of tracer, and thereby no timing issues experienced with the dye used today. This means less preparation time prior to surgery, and reduced risk of lymph edema due to excessive removal of colored lymph nodes. • Improved staging due to truly combined information in a PET/MR system including tracer migration analysis.

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.

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 (99m)Tc. 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 (99m)Tc-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 (99m)Tc-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 (99m)Tc-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 (99m)Tc. The accumulation of (99m)Tc-SPIONs in lymph nodes after subcutaneous injection in animals, verified by SPECT/MRI, is encouraging for applications in breast cancer and malignant melanoma. (Less)

Aim: To investigate the size-dependent lymphatic uptake of nanoparticles in mice with rapidly growing syngeneic tumors. Materials & methods: Mice were inoculated subcutaneously with EL4 lymphoma cells and on day 5 or day 6 of tumor growth, injected peritumorally with either 29 nm or 58 nm of ultra-small superparamagnetic iron oxide nanoparticles. Twenty-four hours later the animals were imaged using MRI. Results & conclusion: The larger of the two particles can only be detected in the lymph node when injected in animals with 6-day-old tumors while the 29 nm ultra-small superparamagnetic iron oxide nanoparticle is observed on both time points. Tumor mass greatly impacts the size of particles that are transported to the lymph nodes., This study aims to improve the way the spreading of certain forms of cancer is detected. The method, known as sentinel lymph node detection, locates and removes the first lymph node that drains the tumor to examine it for cancer cells. The authors want to improve the way this is done by using a new contrast agent based on nanoparticles. Two different sizes of particles were injected around the implanted tumors in mice and imaged with MRI. The results indicate that the mass of the tumor plays a crucial role in the transport of nanoparticles to the lymph node.

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.

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.

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. A protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We have used phage display technology to identify peptides binding to B. pseudomallei protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The cyclic heptapeptides from two of the phage clones (Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys) were identical and showed the strongest phage-protease interaction as detected by ELISA. Four of the five selected phages at the amount of 1013 phages could inhibit B. pseudomallei protease activity by approximately 50%.

EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.