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. The sentinel lymph node (SLN) is defined as the first regional lymph node receiving lymphatic drainage from a malignant tumor (1) and the first node to which metastatic cells are likely to anchor. Therefore, accurate detection and characterization of the SLN is of major importance for cancer staging and for the choice of therapy in patients with breast cancer and malignant melanoma. The current gold standard relies on lymphoscintigraphy after intradermal injection of radiolabeled colloids and blue dye to intraoperatively identify the SLN by dissection and histopathologic examination (2). The radiopharmaceuticals most frequently used for SLN imaging are 99m Tc-labeled colloids and macromolecules such as trisulfide, dextran, and human serum albumin (3-5). The current technique, however, is limited because of the nonspecificity of the tracer and the lack of anatomic information in scintigraphic images. Preoperative planning and identification of the SLN often rely on the experience of the surgeon.We propose combining information from high-resolution MRI and high-sensitivity SPECT images to provide more accurate and less invasive identification of the SLN before surgery. The use of radioactivity would help to ...

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.

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.

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.

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.