Eunice Y. Chen

Clinical EPR: Unique Opportunities and Some Challenges

Electron paramagnetic resonance (EPR) spectroscopy has been well established as a viable technique for measurement of free radicals and oxygen in biological systems, from in vitro cellular systems to in vivo small animal models of disease. However, the use of EPR in human subjects in the clinical setting, although attractive for a variety of important applications such as oxygen measurement, is challenged with several factors including the need for instrumentation customized for human subjects, probe, and regulatory constraints. This article describes the rationale and development of the first clinical EPR systems for two important clinical applications, namely, measurement of tissue oxygen (oximetry) and radiation dose (dosimetry) in humans. The clinical spectrometers operate at 1.2 GHz frequency and use surface-loop resonators capable of providing topical measurements up to 1 cm depth in tissues. Tissue pO2 measurements can be carried out noninvasively and repeatedly after placement of an oxygen-sensitive paramagnetic material (currently India ink) at the site of interest. Our EPR dosimetry system is capable of measuring radiation-induced free radicals in the tooth of irradiated human subjects to determine the exposure dose. These developments offer potential opportunities for clinical dosimetry and oximetry, which include guiding therapy for individual patients with tumors or vascular disease by monitoring of tissue oxygenation. Further work is in progress to translate this unique technology to routine clinical practice.

Advances in Probes and Methods for Clinical EPR Oximetry

EPR oximetry, which enables reliable, accurate, and repeated measurements of the partial pressure of oxygen in tissues, provides a unique opportunity to investigate the role of oxygen in the pathogenesis and treatment of several diseases including cancer, stroke, and heart failure. Building on significant advances in the in vivo application of EPR oximetry for small animal models of disease, we are developing suitable probes and instrumentation required for use in human subjects. Our laboratory has established the feasibility of clinical EPR oximetry in cancer patients using India ink, the only material presently approved for clinical use. We now are developing the next generation of probes, which are both superior in terms of oxygen sensitivity and biocompatibility including an excellent safety profile for use in humans. Further advances include the development of implantable oxygen sensors linked to an external coupling loop for measurements of deep-tissue oxygenations at any depth, overcoming the current limitation of 10 mm. This paper presents an overview of recent developments in our ability to make meaningful measurements of oxygen partial pressures in human subjects under clinical settings.

Skeletal Muscle and Glioma Oxygenation by Carbogen Inhalation in Rats: A Longitudinal Study by EPR Oximetry Using Single-Probe Implantable Oxygen Sensors

The feasibility of EPR oximetry using a single-probe implantable oxygen sensor (ImOS) was tested for repeated measurement of pO₂ in skeletal muscle and ectopic 9L tumors in rats. The ImOS (50 mm length) were constructed using nickel-chromium alloy wires, with lithium phthalocyanine (LiPc, oximetry probe) crystals loaded in the sensor loop and coated with AF 2400(®) Teflon. These ImOS were implanted into the skeletal muscle in the thigh and subcutaneous 9L tumors. Dynamic changes in tissue pO₂ were assessed by EPR oximetry at baseline, during tumor growth, and repeated hyperoxygenation with carbogen breathing. The mean skeletal muscle pO₂ of normal rats was stable and significantly increased during carbogen inhalation in experiments repeated for 12 weeks. The 9L tumors were hypoxic with a tissue pO₂ of 12.8 ± 6.4 mmHg on day 1; however, the response to carbogen inhalation varied among the animals. A significant increase in the glioma pO₂ was observed during carbogen inhalation on day 9 and day 14 only. In summary, EPR oximetry with ImOS allowed direct and longitudinal oxygen measurements in deep muscle tissue and tumors. The heterogeneity of 9L tumors in response to carbogen highlights the need to repeatedly monitor pO₂ to confirm tumor oxygenation so that such changes can be taken into account in planning therapies and interpreting results.

Modulation of hypoxia by magnetic nanoparticle hyperthermia to augment therapeutic index.

A hypoxic microenvironment in solid tumors has been known to cause resistance to standard therapies and to increase the malignant potential of tumors. The utilization of magnetic nanoparticle hyperthermia (mNPH) has shown promise in improving therapeutic outcome by (1) killing of hypoxic tumor cells directly and (2) increasing tumor oxygenation and therefore susceptibility to therapies. In this study, the interaction of a hypoxic microenvironment with mNPH efficacy was investigated in a human breast cancer orthotopic xenograft model. Using electron paramagnetic resonance (EPR) to assess in vivo oxygen concentration in tumors repeatedly and non-invasively, we found that mNPH increased tumor pO₂ from 3.5 to 68.8 mmHg on average for up to 10 days. Tumors treated once with mNPH showed growth delay. On Transmission Electron Microscopy, magnetic nanoparticles (mNPs) were localized intracellularly in multiple vesicles in the cytoplasm of cells within tumors 48 h after incubation of mNP. In conclusion, mNPH increased tumor oxygenation in vivo and resulted in decreased growth of hypoxic tumors. Future studies will establish tumor pO₂-guided multimodal therapies, such as mNPH and radiation, to improve therapeutic efficacy.

Development of the Implantable Resonator System for Clinical EPR Oximetry

Hypoxic tumors are more resistant to radiotherapy and chemotherapy, which decreases the efficacy of these common forms of treatment. We have been developing implantable paramagnetic particulates to measure oxygen in vivo using electron paramagnetic resonance. Once implanted, oxygen can be measured repeatedly and non-invasively in superficial tissues (<3 cm deep), using an electron paramagnetic resonance spectrometer and an external surface-loop resonator. To significantly extend the clinical applications of electron paramagnetic resonance oximetry, we developed an implantable resonator system to obtain measurements at deeper sites. This system has been used to successfully obtain oxygen measurements in animal studies for several years. We report here on recent developments needed to meet the regulatory requirements to make this technology available for clinical use. radio frequency heating is discussed and magnetic resonance compatibility testing of the device has been carried out by a Good Laboratory Practice-certified laboratory. The geometry of the implantable resonator has been modified to meet our focused goal of verifying safety and efficacy for the proposed use of intracranial measurements and also for future use in tissue sites other than the brain. We have encapsulated the device within a smooth cylindrical-shaped silicone elastomer to prevent tissues from adhering to the device and to limit perturbation of tissue during implantation and removal. We have modified the configuration for simultaneously measuring oxygen at multiple sites by developing a linear array of oxygen sensing probes, which each provide independent measurements. If positive results are obtained in additional studies which evaluate biocompatibility and chemical characterization, we believe the implantable resonator will be at a suitable stage for initial testing in human subjects.

Skeletal Muscle Oxygenation Measured by EPR Oximetry Using a Highly Sensitive Polymer-Encapsulated Paramagnetic Sensor.

We have incorporated LiNc-BuO, an oxygen-sensing paramagnetic material, in polydimethylsiloxane (PDMS), which is an oxygen-permeable, biocompatible, and stable polymer. We fabricated implantable and retrievable oxygen-sensing chips (40 % LiNc-BuO in PDMS) using a 20-G Teflon tubing to mold the chips into variable shapes and sizes for in vivo studies in rats. In vitro EPR measurements were used to test the chips oxygen response. Oxygen induced linear and reproducible line broadening with increasing partial pressure (pO2). The oxygen response was similar to that of bare (unencapsulated) crystals and did not change significantly on sterilization by autoclaving. The chips were implanted in rat femoris muscle and EPR oximetry was performed repeatedly (weekly) for 12 weeks post-implantation. The measurements showed good reliability and reproducibility over the period of testing. These results demonstrated that the new formulation of OxyChip with 40 % LiNc-BuO will enable the applicability of EPR oximetry for long-term measurement of oxygen concentration in tissues and has the potential for clinical applications.

Development of a biodegradable iron oxide nanoparticle gel for tumor bed therapy.

Treatments of the post-operative surgical bed have proven appealing as the majority of cancer recurrence following tumor resection occurs at the tumor margin. A novel, biodegradable pullulan-based gel infused with magnetic iron oxide nanoparticles (IONP) is presented here for surgical bed administration followed by hyperthermia therapy via alternating magnetic field (AMF) activation. Pullulan is a water soluble, film-forming starch polymer that degrades at the postoperative wound site to deliver the IONP payload, targeting the remaining cancer cells. Different gel formulations containing various % wt of pullulan were tested for IONP elution. Elution levels and amount of gel degradation were measured by immersing the gel in de-ionized water for one hour then measuring particle concentrations in the supernatant and the mass of the remaining gel formulation. The most promising gel formulations will be tested in a murine model of surgical bed resection to assess in vivo gel dissolution, IONP cell uptake kinetics via histology and TEM analysis, and heating capability of the gel with AMF exposure.

Paired-agent imaging for resection during surgery (PAIRS) of head and neck squamous cell carcinomas(Conference Presentation)

Ninety percent of patients with head and neck squamous cell carcinomas (HNSCC) have overexpression of epidermal growth factor receptor (EGFR), which is correlated with poor prognosis. Complete surgical resection of HNSCC tumors has a large impact on patient survival, where detection of tumor at or close to surgical margins increases the risk of death at 5-years by 90%. In addition, large surgical margins can greatly increase the morbidity experienced by the patient due to functional and cosmetic damage of oral and facial structures. Single fluorescence targeting agents are often used for tumor detection in in vivo pre-clinical imaging; however, the arising signal is qualitative at best because it is a complex mixture of vascular perfusion, vascular leakage, inhibited lymphatic clearance, and receptor binding. In vivo ratiometric receptor concentration imaging (RCI) allows quantification of receptor expression (hence identification of cancerous tissue) by utilizing co-administered paired-agents consisting of a targeted agent and non-targeted perfusion agent to reference the plasma delivery and leakage. A panel of HNSCC tumors with varying levels of EGFR expression (SCC-15 >SCC-25 > SCC-09) have been imaged using ABY-029, a clinically relevant anti-EGFR affibody labeled with IRDye 800CW, and affibody control imaging agent labeled with IRDye 680RD. RCI maps of in vivo tissue have been created and are spatially correlated with EGFR and CD31 immunohistochemistry and basic H and E staining. The RCI threshold parameters for distinguishing tumor from normal tissues (skin and muscle) and the accuracy of margin detection in these tumors will be presented. RCI surgical resection will be further developed using a novel multi-channel, gated fluorescence-guided surgery (FGS) imaging system that is capable of performing RCI in normal room light.

Oxygen microenvironment affects the uptake of nanoparticles in head and neck tumor cells

Survival of head and neck cancer patients has not improved in several decades despite advances in diagnostic and therapeutic techniques. Tumor hypoxia in head and neck cancers is a critical factor that leads to poor prognosis, resistance to radiation and chemotherapies, and increased metastatic potential. Magnetic nanoparticle hyperthermia (mNPHT) is a promising therapy for hypoxic tumors because nanoparticles (NP) can be directly injected into, or targeted to, hypoxic tumor cells and exposed to alternating magnetic fields (AMF) to induce hyperthermia. Magnetic NPHT can improve therapeutic effectiveness by two modes of action: 1) direct killing of hypoxic tumor cells; and 2) increase in tumor oxygenation, which has the potential to make the tumor more susceptible to adjuvant therapies such as radiation and chemotherapy. Prior studies in breast cancer cells demonstrated that a hypoxic microenvironment diminished NP uptake in vitro; however, mNPHT with intratumoral NP injection in hypoxic tumors increased tumor oxygenation and delayed tumor growth. In this study, head and neck squamous cell carcinoma (HNSCC) cell lines were incubated in normoxic, hypoxic, and hyperoxic conditions with iron oxide NP for 4-72 hours. After incubation, the cells were analyzed for iron uptake by mass spectrometry, Prussian blue staining, and electron microscopy. In contrast to breast cancer cells, uptake of NPs was increased in hypoxic microenvironments as compared to normoxic conditions in HNSCC cells. In future studies, we will confirm the effect of the oxygen microenvironment on NP uptake and efficacy of mNPHT both in vitro and in vivo.