Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O
) delivery is ...limited to slow passive diffusion from transplantation sites through the poorly O
-soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O
delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O
diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O
transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months.
Abstract
Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in ...curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO
2
) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R
1
) of oxygen-sensitive spin probes such as trityl OX071 and pO
2
to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO
2
measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO
2
calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO
2
measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO
2
imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.
Purpose
Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid ...tumors are known to have hypoxia or low oxygen levels (pO
2
≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO
2
). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging.
Methods
A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 μL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries.
Results
For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol.
Conclusions
This study demonstrates the performance of the world’s first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications.
Purpose
Progress toward developing a novel radiocontrast agent for determining pO
2
in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic ...resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue.
Procedures
The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10–20 nm) polymer layer to separate the imaging agent and body environment while still allowing O
2
to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense.
Results
Side-by-side
in vivo
imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO
2
maps of solid tumors
Conclusions
A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality.
This study investigated the location and distribution of paramagnetic species in apple seeds using electron paramagnetic resonance (EPR) and X-band (9 GHz) EPR imaging (EPRI). EPR primarily detected ...two paramagnetic species per measured seed. These two different radical species were assigned as stable radicals and Mn2+ species based on the g values and hyperfine components. The signal from the stable radical was noted at g ≈ 2.00 and was strong and relatively stable. The subsequent noninvasive EPRI of the radical present in each seed revealed that the stable radicals were located primarily in the seed coat, with very few radicals observed in the cotyledon of the seed. These results indicate that the stable radical species were only found within the seed coat, and few radical species were found in other seed parts.
Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose ...radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.
Yakov Sergeevich Lebedev was a pioneer in high-frequency EPR, taking advantage of the separation of
g
-factor anisotropy effects from nuclear hyperfine splitting and the higher-frequency molecular ...motion sensitivity from higher-frequency measurements (Appl Magn Reson 7: 339–362, 1994). This article celebrates a second EPR subfield in which Prof. Lebedev pioneered, EPR imaging (Chem Phys Lett 99: 301–304, 1983). We celebrate the clinical enhancements that are suggested in this low-frequency work and imaging application to animal physiology at lower-than-standard EPR frequencies.
Purpose
Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A ...major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response.
Procedures
Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV’s effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival.
Results
Hypoxic sarcomas were more radioresistant than normoxic sarcomas (
p
=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (
p
=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV.
Conclusion
EPROI provides 3D high-resolution pO
2
quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.
Purpose
This study aimed to develop a biocompatible oximetric electron paramagnetic resonance (EPR) spin probe with reduced self-relaxation, and sensitivity to oxygen for a higher signal-to-noise ...ratio and longer relaxation times at high oxygen concentration, compared to the reference spin probe OX071.
Procedures
SOX71 was synthesized by succinylation of the twelve alcohol groups of OX071 spin probe and characterized by EPR at X-Band (9.5 GHz) and at low field (720 MHz). The biocompatibility of SOX71 was tested
in vitro
and
in vivo
in mice. A pharmacokinetic study was performed to determine the best time frame for EPR imaging. Finally, a proof-of-concept EPR oxygen imaging was performed on a mouse model of a fibrosarcoma tumor.
Results
SOX71 was synthesized in one step from OX071. SOX71 exhibits a narrow line EPR spectrum with a peak-to-peak linewidth of 66 mG, similar to OX071. SOX71 does not bind to albumin nor show cell toxicity for the concentrations tested up to 5 mM. No toxicity was observed after systemic delivery via intraperitoneal injection in mice at twice the dose required for EPR imaging. After the injection, the probe is readily absorbed into the bloodstream, with a peak blood concentration half an hour, post-injection. Then, the probe is quickly cleared by the kidney with a half-life of ~ 45 min. SOX71 shows long relaxation times under anoxic condition (T
1e
= 9.5 µs and T
2e
= 5.1 µs; SOX71 = 1 mM in PBS at 37 °C,
p
O
2
= 0 mmHg, 720 MHz). Both the relaxation rates R
1e
and R
2e
show a decreased sensitivity to
p
O
2,
leading to twice longer relaxation times under room air conditions (
p
O
2
= 159 mmHg) compared to OX071. This is ideal for oxygen imaging in samples with a wide range of
p
O
2
. Both the relaxation rates R
1e
and R
2e
show a decreased sensitivity to self-relaxation compared to OX071, with a negligible effect of the probe concentration on R
1e
. SOX71 was successfully applied to image oxygen in a tumor.
Conclusion
SOX71, a succinylated derivative of OX071 was synthesized, characterized, and applied for
in vivo
EPR tumor oxygen imaging. SOX71 is highly biocompatible, and shows decreased sensitivity to oxygen and self-relaxation. This first report suggests that SOX71 is superior to OX071 for absolute oxygen mapping under a broad range of
p
O
2
values.
Spectral–spatial images of lithium phthalocyanine (LiPC) phantom (left-hand) and a pepper seed (right-hand). Display omitted
•Locations of radical species in black pepper seeds were investigated by ...continuous wave (CW) EPR and 9GHz EPR imaging.•Lithium phthalocyanine (LiPC) phantom was used to examine 9GHz EPR imaging capabilities.•Radical species were mostly located at the seed surface.•CW EPR and EPR imaging were useful for determination of the spatial distribution of paramagnetic species in various seeds.
In this study, noninvasive 9GHz electron paramagnetic resonance (EPR)-imaging and continuous wave (CW) EPR were used to investigate the locations of paramagnetic species in black pepper seeds without further irradiation. First, lithium phthalocyanine (LiPC) phantom was used to examine 9GHz EPR imaging capabilities. The 9GHz EPR-imager easily resolved the LiPC samples at a distance of ∼2mm. Then, commercially available black pepper seeds were measured. We observed signatures from three different radical species, which were assigned to stable organic radicals, Fe3+, and Mn2+ complexes. In addition, no EPR spectral change in the seed was observed after it was submerged in distilled H2O for 1h. The EPR and spectral–spatial EPR imaging results suggested that the three paramagnetic species were mostly located at the seed surface. Fewer radicals were found inside the seed. We demonstrated that the CW EPR and 9GHz EPR imaging were useful for the determination of the spatial distribution of paramagnetic species in various seeds.
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