Multimodal sentinel lymph node imaging using nanoscale-tailored particles; The importance of particle size

Abstract: As a cancer grows it will, at a certain stage of disease progression, start spreading in the body. For several types of cancer this spreading will first occur through the lymphatic system and the first place where metastatic cell will be encountered is the first lymph node that drains the area where the tumor is located. This lymph node is defined as the sentinel lymph node (SLN). By analyzing the SLN for the presence of tumor cells the metastatic status of the primary tumor can be determined. Today the process of sentinel lymph node biopsy is lengthy and requires numerous steps to be taken in succession. A major reason for this is the contrast agents that are used for the procedure. Exchanging these contrast agents for a more suitable compound could streamline the procedure allowing for a higher throughput of patients as well as eliminating the negative effects associated with the current contrast agents.
In this thesis the possibility of developing a novel multimodal contrast agent for sentinel lymph node biopsies, based on ultra-small superparamagnetic iron oxide nanoparticles (USPIOs), is explored. The USPIOs used here are non-toxic in the applicable amounts and are coated with a polyethylene glycol based substance. The coating material gives the particles the ability for conjugation of functional surface groups through rather simple chemistry. The configuration of the coating also acts as a natural chelate, allowing for the attachment of metallic ion based radioisotopes.
Emphasis has been put on investigating the multimodal traits of differently sized USPIOs as well as their retention in SLN of healthy animals and a disease model.
In healthy rats it was concluded that 15 nm USPIOs accumulate faster and at a higher amount in the popliteal lymph node, than 27 or 58 nm USPIOs, after subcutaneous injection in the hind paw. It was also documented that the USPIOs were readily detectable with magnetic resonance imaging (MRI) and with fluorescence microscopy. The multimodal nature of the nanoparticles was further evaluated by detecting injected 31 nm and 67 nm USPIOs in healthy rats with magnetomotive ultrasound. The results showed that the smaller of the two sizes of particles were present in the SLN at higher amount than the larger USPIOs. It also demonstrated that the magnetomotive ultrasound technique has potential as an intraoperative tool for locating nanoparticle-laden lymph nodes.
The lymphatic retention of two different sizes of USPIOs, 29 nm and 58 nm, were tested in a subcutaneous EL-4 tumor model in mice. The particles were injected peritumorally and the results were detected with MRI in vivo and fluorescence imaging ex vivo. As the rapidly growing EL-4 tumors increased in size the dynamics of lymphatic uptake of USPIOs was altered. In animals injected with 58 nm nanoparticles on day-5 of tumor growth, only 1 of 5 had detectable amount of particles in the SLN when imaged with MRI the next day. The corresponding findings for the 29 nm USPIOs were 4 of 5 SLN with detectable amounts of nanoparticles. When the particles were injected in animals with 6 days old tumors USPIOs could be detected in all SLN the following day, regardless of particle size.
The distribution of the two different sizes of USPIOs was further evaluated by labeling the particles with radionuclides. The two particle sizes were labeled with either Indium-111 (26 nm) or Lutetium-177 (64 nm). The labeled USPIOs were mixed and injected peritumorally in EL-4 tumor bearing mice. The results were analyzed with SPECT/CT and autoradiography and demonstrated the possibility to detect and separate the two sizes of nanoparticles in vivo with SPECT and ex vivo with autoradiography.
In summary, the results presented in this thesis show that the nanoparticles produced have the potential for truly multimodal imaging. A size dependency for lymphatic uptake of USPIOs could be demonstrated, both in healthy and diseased animals. A size dependent lymphatic uptake of USPIOs could also be related to size of the tumors.