Measurement of Tissue Oxygen as a Novel Approach to Optimizing Red Blood Cell Quality Assessment.

The effectiveness of blood transfusions can be impacted by storage and extensive processing techniques that involve treatment of red blood cells (RBCs) with pathogen reduction technologies (e.g., UV-light and chemical treatment), ex vivo stem cell derivation/maturation methods, and bioengineering of RBCs using nanotechnology. Therefore, there is a need to have methods that assess the evaluation of the effectiveness of transfusions to achieve their intended purpose: to increase oxygenation of critical tissues. Consequently, there has been intense interest in the development of techniques targeted at optimizing the assessment of RBC quality in preclinical and clinical settings. We provide a critical assessment of the ability of currently used methods to provide unambiguous information on oxygen levels in tissues and conclude that they cannot do this. This is because they are based on surrogates for the true goal of transfusion, which is to increase oxygenation of critical organs. This does not mean that they are valueless, but it does indicate that other methods are needed to provide direct measurements of oxygen in tissues. We report here on the initial results of a method that can provide direct assessment of the impact of the transfusion on tissue oxygen: EPR oximetry. It has the potential to provide such information in both preclinical and clinical settings for the assessment of blood quality posttransfusion.

Factors determining barrier properties to oxygen transport across model and cell plasma membranes based on EPR spin-label oximetry.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

Clinical and Statistical Considerations when Assessing Oxygen Levels in Tumors: Illustrative Results from Clinical EPR Oximetry Studies.

The success of treatment for malignancies, especially those undergoing radiation therapy or chemotherapy, has long been recognized to depend on the degree of hypoxia in the tumor. In addition to the prognostic value of knowing the tumor's initial level of hypoxia, assessing the tumor oxygenation during standard therapy or oxygen-related treatments (such as breathing oxygen-enriched gas mixtures or taking drugs that can increase oxygen supply to tissues) can provide valuable data to improve the efficacy of treatments. A series of early clinical studies of tumors in humans are ongoing at Dartmouth and Emory using electron paramagnetic resonance (EPR) oximetry to assess tumor oxygenation, initially and over time during either natural disease progression or treatment. This approach has the potential for reaching the long-sought goal of enhancing the effectiveness of cancer therapy. In order to effectively reach this goal, we consider the validity of the practical and statistical assumptions when interpreting the measurements made in vivo for patients undergoing treatment for cancer.

Impact of myo-inositol trispyrophosphate (ITPP) on tumour oxygenation and response to irradiation in rodent tumour models.

Tumour hypoxia is a well-established factor of resistance in radiation therapy (RT). Myo-inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen-binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO2 upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L-glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L-gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L-gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L-gliomas but was absent in the rhabdomyosarcomas.

Wireless implantable coil with parametric amplification for in vivo electron paramagnetic resonance oximetric applications.

PURPOSE: To develop an implantable wireless coil with parametric amplification capabilities for time-domain electron paramagnetic resonance (EPR) spectroscopy operating at 300 MHz. METHODS: The wireless coil and lithium phthalocyanine (LiPc), a solid paramagnetic probe, were each embedded individually in a biocompatible polymer polydimethoxysiloxane (PDMS). EPR signals from the LiPc embedded in PDMS (LiPc/PDMS) were generated by a transmit-receive surface coil tuned to 300 MHz. Parametric amplification was configured with an external pumping coil tuned to 600 MHz and placed between the surface coil resonator and the wireless coil. RESULTS: Phantom studies showed significant enhancement in signal to noise using the pumping coil. However, no influence of the pumping coil on the oxygen-dependent EPR spectral linewidth of LiPc/PDMS was observed, suggesting the validity of parametric amplification of EPR signals for oximetry by implantation of the encapsulated wireless coil and LiPc/PDMS in deep regions of live objects. In vivo studies demonstrate the feasibility of this approach to longitudinally monitor tissue pO2 in vivo and also monitor acute changes in response to pharmacologic challenges. The encapsulated wireless coil and LiPc/PDMS engendered no host immune response when implanted for ∼3 weeks and were found to be well tolerated. CONCLUSIONS: This approach may find applications for monitoring tissue oxygenation to better understand the pathophysiology associated with wound healing, organ transplantation, and ischemic diseases.

EPR-based oximetric imaging: a combination of single point-based spatial encoding and T1 weighting.

PURPOSE: Spin-lattice relaxation rate (R1 )-based time-domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl-based Oxo71. METHODS: The R1 dependence of the trityl probe Oxo71 on partial oxygen pressure (pO2 ) was assessed using single-point imaging mode of spatial encoding combined with rapid repetition, similar to T1 -weighted MRI, for which R1 was determined from 22 repetition times ranging from 2.1 to 40.0 µs at 300 MHz. The pO2 maps of a phantom with 3 tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R1 and apparent spin-spin relaxation rate ( R2*) simultaneously. RESULTS: The pO2 maps derived from R1 and R2* agreed with the known pO2 levels in the tubes of Oxo71. However, the histograms of pO2 revealed that R1 offers better pO2 resolution than R2* in low pO2 regions. The SDs of pixels at 2% pO2 (15.2 mmHg) were about 5 times lower in R1 -based estimation than R2*-based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO2 map obtained from R1 -based assessment displayed a homogeneous profile in low pO2 regions in tumor xenografts, consistent with previous reports on R2*-based oximetric imaging. The scan time to obtain the R1 map can be significantly reduced using 3 repetition times ranging from 4.0 to 12.0 µs. CONCLUSION: Using the single-point imaging modality, R1 -based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated.

Adipose tissue-derived stem cells in a fibrin implant enhance neovascularization in a peritoneal grafting site: a potential way to improve ovarian tissue transplantation.

STUDY QUESTION: Do two different concentrations of human adipose tissue-derived stem cells (ASCs) embedded inside a fibrin scaffold have the potential to differentiate into vessels and aid vascularization in a peritoneal grafting site intended for ovarian tissue transplantation? SUMMARY ANSWER: Human ASCs in low and high concentrations differentiated into vessels when transplanted to mouse peritoneum inside a fibrin matrix, but only high ASC concentrations significantly increased human vessel area 14 days after transplantation. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, including proangiogenic properties and have been used in tissue engineering to enhance vascularization in transplanted tissues. Fibrin has been studied and used as an ASC-compatible biomaterial. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 22 severe combined immunodeficient mice. In total, 16 mice (eight per group) were intraperitoneally grafted with a fibrin scaffold loaded with two different human ASC concentrations (either 150 000 [L-ASC] or 1 500 000 [H-ASC] cells) and lithium phthalocyanine (LiPc) crystals as oxygen-sensitive probes. Six mice were grafted with an empty fibrin (EF) implant containing only LiPc and served as controls. Levels of partial pressure of oxygen (pO2) in implants were monitored in vivo by electron paramagnetic resonance oximetry (EPR). ASC identification, proliferation, and host and human vascularization were analyzed by immunohistochemistry (IHC). All analyses were performed on post-grafting Days 3, 7 and 14. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), as well as histological (hematoxylin-eosin staining) and IHC (anti-human vimentin, anti-human Ki67, anti-mouse and human double CD34) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 in implants was observed in all groups between Days 3 and 7 (P < 0.001). ASC-loaded implants displayed a tendency towards increased pO2 levels from Days 7 to 14, not observed in EF implants. ASC-loaded implants showed differentiation into human CD34-positive vessels. Total CD34-positive endothelial area was correlated to pO2 values obtained by EPR oximetry (r = 0.6506, P = 0.0019). In the H-ASC group, a greater human CD34-positive vascular surface area was found compared to the L-ASC group 14 days after transplantation (P < 0.0049). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, ASCs transplanted inside a fibrin matrix can differentiate into CD34-positive human vessels. However, other possible mechanisms involved in ASC angiogenic behavior remain to be investigated. WIDER IMPLICATIONS OF THE FINDINGS: High concentrations of ASCs loaded inside a fibrin scaffold could serve as a substrate to prepare a peritoneal grafting site over 14 days, in order to enhance vascularization once human ovarian tissue is grafted. Our proposed preparation of the grafting site would not only benefit ovarian tissue transplantation, but also other experimental avascular grafting procedures. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, Télévie Grant no. 7.6515.16F awarded to DDM and Grant 5/4/150/5 awarded to M.M.D. [CAA is FRS-FNRS research associate]), Fonds Spéciaux de Recherche, and Fondation St Luc, Foundation Against Cancer, and donations from the Ferrero family. None of the authors have any competing interests to declare.

Effect of amifostine, a radiation-protecting drug, on oxygen concentration in tissue measured by EPR oximetry and imaging.

Effect of amifostine, a radiation-protecting drug, on muscle tissue partial pressure of oxygen was investigated by electron paramagnetic resonance spectroscopy and imaging. When amifostine was administered intraperitoneally or intravenously to mice, the linewidth of the electron paramagnetic resonance spectra of the lithium octa-n-butoxy-substituted naphthalocyanine implanted in the mouse leg muscle decreased. Electron paramagnetic resonance oximetry using a lithium octa-n-butoxy-substituted naphthalocyanine probe and electron paramagnetic resonance oxygen mapping using a triarylmethyl radical paramagnetic probe was useful to quantify pressure of oxygen in the tissues of living mice. The result of electron paramagnetic resonance oximetric imaging showed that administration of amifostine could decrease pressure of oxygen in the muscle and also tumor tissues. This finding suggests that lowering pressure of oxygen in tissues might contribute in part to the radioprotection of amifostine.

Functional electron paramagnetic resonance imaging of ischemic rat heart: Monitoring of tissue oxygenation and pH.

PURPOSE: Electron paramagnetic resonance (EPR) imaging in the spectral-spatial domain with application of soluble paramagnetic probes provides an opportunity for spatially resolved functional measurements of living objects. The purpose of this study was to develop EPR methods for visualization of oxygenation and acidosis of ischemic myocardium. METHODS: EPR oxygen measurements were performed using isotopically substituted (2) H,(15) N-dicarboxyproxyl. The radical has an EPR line width of 320 mG and oxygen-induced line broadening of 0.53 mG/mm Hg, providing oxygen sensitivity down to 5 µM. pH measurements were performed using previously developed pH-sensitive imidazoline nitroxide. The radical has an EPR spectrum with pH-dependable hyperfine splitting, pK = 6.6, providing pH sensitivity of approximately 0.05 U in the physiological range. RESULTS: EPR imaging of isolated and perfused rat hearts was performed in the two-dimensional + spectral domain. The spatial resolution of the measurements was about 1.4 mm. Marked tissue hypoxia was observed in the ischemic area of the heart after occlusion of the left anterior descending coronary artery. Tissue oxygenation was partly restored upon reperfusion. EPR mapping of myocardial pH indicated acidosis of the ischemic area down to pH 6.7-6.8. CONCLUSION: This study demonstrates the capability of low-field EPR and the nitroxide spin probes for mapping of myocardial oxygenation and pH. The developed approaches might be used for noninvasive investigation of microenvironment on living objects. Magn Reson Med 76:350-358, 2016. © 2015 Wiley Periodicals, Inc.

Nanofiber-based paramagnetic probes for rapid, real-time biomedical oximetry.

EPR (electron paramagnetic resonance) based biological oximetry is a powerful tool that accurately and repeatedly measures tissue oxygen levels. In vivo determination of oxygen in tissues is crucial for the diagnosis and treatment of a number of diseases. Here, we report the first successful fabrication and remarkable properties of nanofiber sensors for EPR-oximetry applications. Lithium octa-n-butoxynaphthalocyanine (LiNc- BuO), an excellent paramagnetic oxygen sensor, was successfully encapsulated in 300-500 nm diameter fibers consisting of a core of polydimethylsiloxane (PDMS) and a shell of polycaprolactone (PCL) by electrospinning. This core-shell nanosensor (LiNc-BuO-PDMS-PCL) shows a linear dependence of linewidth versus oxygen partial pressure (pO2). The nanofiber sensors have response and recovery times of 0.35 s and 0.55 s, respectively, these response and recovery times are ~12 times and ~218 times faster than those previously reported for PDMS-LiNc-BuO chip sensors. This greater responsiveness is likely due to the high porosity and excellent oxygen permeability of the nanofibers. Electrospinning of the structurally flexible PDMS enabled the fabrication of fibers having tailored spin densities. Core-shell encapsulation ensures the non-exposure of embedded LiNc-BuO and mitigates potential biocompatibility concerns. In vitro evaluation of the fiber performed under exposure to cultured cells showed that it is both stable and biocompatible. The unique combination of biocompatibility due to the PCL 'shell,' the excellent oxygen transparency of the PDMS core, and the excellent oxygen-sensing properties of LiNc-BuO makes LiNc-BuO-PDMS-PCL platform promising for long-term oximetry and repetitive oxygen measurements in both biological systems and clinical applications.

Trityl radicals in perfluorocarbon emulsions as stable, sensitive, and biocompatible oximetry probes.

EPR oximetry with the use of trityl radicals can enable sensitive O2 measurement in biological cells and tissues. However, in vitro cellular and in vivo biological applications are limited by rapid trityl probe degradation or biological clearance and the need to enhance probe O2 sensitivity. We synthesized novel perfluorocarbon (PFC) emulsions, ∼200nm droplet size, containing esterified perchlorinated triphenyl methyl (PTM) radicals dispersed in physiological aqueous buffers. These formulations exhibit excellent EPR signal stability, over 20-fold greater than free PTM probes, with high oxygen sensitivity ∼17mG/mmHg enabling pO2 measurement in aqueous solutions or cell suspensions with sensitivity >0.5mmHg. Thus, PFC-PTM probes hold great promise to enable combined O2 delivery and sensing as needed to restore or enhance tissue oxygenation in disease.

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 chip's 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.

Monitoring oxygen levels in orthotopic human glioma xenograft following carbogen inhalation and chemotherapy by implantable resonator-based oximetry.

Hypoxia is a critical hallmark of glioma, and significantly compromises treatment efficacy. Unfortunately, techniques for monitoring glioma pO2 to facilitate translational research are lacking. Furthermore, poor prognosis of patients with malignant glioma, in particular glioblastoma multiforme, warrant effective strategies that can inhibit hypoxia and improve treatment outcome. EPR oximetry using implantable resonators was implemented for monitoring pO2 in normal cerebral tissue and U251 glioma in mice. Breathing carbogen (95% O2 + 5% CO2 ) was tested for hyperoxia in the normal brain and glioma xenografts. A new strategy to inhibit glioma growth by rationally combining gemcitabine and MK-8776, a cell cycle checkpoint inhibitor, was also investigated. The mean pO2 of left and right hemisphere were ∼56-69 mmHg in the normal cerebral tissue of mice. The mean baseline pO2 of U251 glioma on the first and fifth day of measurement was 21.9 ± 3.7 and 14.1 ± 2.4 mmHg, respectively. The mean brain pO2 including glioma increased by at least 100% on carbogen inhalation, although the response varied between the animals over days. Treatment with gemcitabine + MK-8776 significantly increased pO2 and inhibited glioma growth assessed by MRI. In conclusion, EPR oximetry with implantable resonators can be used to monitor the efficacy of carbogen inhalation and chemotherapy on orthotopic glioma in mice. The increase in glioma pO2 of mice breathing carbogen can be used to improve treatment outcome. The treatment with gemcitabine + MK-8776 is a promising strategy that warrants further investigation.

In vivo evidence of methamphetamine induced attenuation of brain tissue oxygenation as measured by EPR oximetry.

Abuse of methamphetamine (METH) is a major and significant societal problem in the US, as a number of studies have suggested that METH is associated with increased cerebrovascular events, hemorrhage or vasospasm. Although cellular and molecular mechanisms involved in METH-induced toxicity are not completely understood, changes in brain O2 may play an important role and contribute to METH-induced neurotoxicity including dopaminergic receptor degradation. Given that O2 is the terminal electron acceptor for many enzymes that are important in brain function, the impact of METH on brain tissue pO2 in vivo remains largely uncharacterized. This study investigated striatal tissue pO2 changes in male C57BL/6 mice (16-20 g) following METH administration using EPR oximetry, a highly sensitive modality to measure pO2 in vivo, in situ and in real time. We demonstrate that 20 min after a single injection of METH (8 mg/kg i.v.), the striatal pO2 was reduced to 81% of the pretreatment level and exposure to METH for 3 consecutive days further attenuated striatal pO2 to 64%. More importantly, pO2 did not recover fully to control levels even 24 h after administration of a single dose of METH and continual exposure to METH exacerbates the condition. We also show a reduction in cerebral blood flow associated with a decreased brain pO2 indicating an ischemic condition. Our findings suggests that administration of METH can attenuate brain tissue pO2, which may lead to hypoxic insult, thus a risk factor for METH-induced brain injury and the development of stroke in young adults.

Increasing oxygen tension in tumor tissue using ultrasound sensitive O2 microbubbles.

Low tissue oxygenation significantly impairs the effectiveness of cancer therapy and promotes a more aggressive phenotype. Many strategies to improve tissue oxygenation have been proposed throughout the years, but only a few showed significant effects in clinical settings. We investigated stability and ultrasound pulse (UP) triggered oxygen release from phospholipid coated oxygen microbubbles (OMB) in vitro and in murine tumors in vivo using EPR oximetry. In solution, the investigated microbubbles are stable and responsive to ultrasound pulse. The addition of the OMB solution alone resulted in an increase in pO2 of approximately 70 mmHg which was further increased for an additional 80 mmHg after the application of UP. The in vivo kinetic study revealed a substantial, up to 120 mmHg, increase in tumor pO2 after UP application and then pO2 was decreasing for 20 min for intravenous injection and 15 min for intratumoral injection. A significant increase was also observed in groups that received microbubbles filled with nitrogen and ultrasound pulse and OMB without UP, but the effect was much lower. Oxygen microbubbles lead to a decrease in HIF-1a and VEGF-A both at the level of mRNA and protein. Toxicity analysis showed that intravenous injection of OMB does not cause oxidative damage to the heart, liver, or kidneys. However, elevated levels of oxidative damage to lipids and proteins were observed short-term in tumor tissue. In conclusion, we have demonstrated the feasibility of oxygen microbubbles in delivering oxygen effectively and safely to the tumor in living animals. Such treatment might enhance the effectiveness of other anticancer therapies.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

EPR Oximetry Sensor-Developing a TAM Derivative for In Vivo Studies.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.

Spheroid Size Does not Impact Metabolism of the ß-blocker Propranolol in 3D Intestinal Fish Model.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Hyperoxygenation as a Therapeutic Supplement for Treatment of Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) refers to a group of biologically aggressive breast cancers that do not express estrogen, progesterone or epidermal growth factor receptor 2 hormone receptors. Each subset of TNBC has a unique molecular profile and may require specific treatments. A combination of surgery and chemotherapy followed by radiation therapy is the standard treatment mode for TNBC patients. Tumor oxygen status (hypoxia) is a key factor that may compromise the effectiveness of radiation treatment, as it is known that hypoxia can confer radiation resistance. In this study, we characterized MDA-MB-231 orthotropic xenograft tumors with respect to tumor oxygen level and their response to supplemental oxygen therapy in combination with paclitaxel and radiation therapy. We observed that the TNBC tumors became severely hypoxic (pO2 < 4 mmHg) within 1 week of tumor growth and responded poorly to administration of respiratory hyperoxygenation (100% O2) to mitigate hypoxia. However, periodic administration of supplemental oxygen (100% O2; 60 min/day for 21 days) showed a significant inhibitory effect on tumor volume when compared to control (1,023 ± 32 mm3 vs. 1,378 ± 114 mm3; p < 0.05). Combination of supplemental oxygen with paclitaxel and radiation therapy led to a significant reduction in tumor growth when compared to radiation alone (239 ± 40 mm3 vs. 390 ± 32 mm3; p < 0.05). The therapeutic enhancement by supplemental oxygen is possibly attributed to increase in tumor oxygenation with paclitaxel at the time of radiation treatment. These findings may have important implications in the understanding of the role of oxygen and supplemental oxygen therapy for the treatment of TNBC patients.