Ramasamy P. Pandian

Novel particulate spin probe for targeted determination of oxygen in cells and tissues.

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.

Endothelium-Derived Nitric Oxide Regulates Postischemic Myocardial Oxygenation and Oxygen Consumption by Modulation of Mitochondrial Electron Transport

Background—Nitric oxide (NO) production is increased in postischemic myocardium, and NO can control mitochondrial oxygen consumption in vitro. Therefore, we investigated the role of endothelial NO synthase (eNOS)–derived NO on in vivo regulation of oxygen consumption in the postischemic heart. Methods and Results—Mice were subjected to 30 minutes of coronary ligation followed by 60 minutes of reperfusion. Myocardial oxygen tension (Po2) was monitored by electron paramagnetic resonance oximetry. In wild-type, N-nitro-l-arginine methyl ester (L-NAME)–treated (with 1 mg/mL in drinking water), and eNOS knockout (eNOS−/−) mice, no difference was observed among baseline myocardial Po2 values (8.6±0.7, 10.0±1.2, and 10.1±1.2 mm Hg, respectively) or those measured at 30 minutes of ischemia (1.4±0.6, 2.3±0.9, and 3.1±1.4 mm Hg, respectively). After reperfusion, myocardial Po2 increased markedly (P<0.001 versus baseline in each group) but was much lower in L-NAME–treated and eNOS−/− mice (17.4±1.6 and 20.4±1.9 mm Hg) than in wild-type mice (46.5±1.7 mm Hg; P<0.001). A transient peak of myocardial Po2 was observed at early reperfusion in wild-type mice. No reactive hyperemia was observed during early reperfusion. Endothelial NO decreased the rate-pressure product (P<0.05), upregulated cytochrome c oxidase (CcO) mRNA expression (P<0.01) with no change in CcO activity, and inhibited NADH dehydrogenase (NADH-DH) activity (P<0.01) without alteration of NADH-DH mRNA expression. Peroxynitrite-mediated tyrosine nitration was higher in hearts from wild-type mice than in eNOS−/− or L-NAME–treated hearts. Conclusions—eNOS-derived NO markedly suppresses in vivo O2 consumption in the postischemic heart through modulation of mitochondrial respiration based on alterations in enzyme activity and mRNA expression of NADH-DH and CcO. The marked myocardial hyperoxygenation in reperfused myocardium may be a critical factor that triggers postischemic remodeling.

Simultaneous Measurement of Oxygenation in Intracellular and Extracellular Compartments of Lung Microvascular Endothelial Cells

A new technique is described for simultaneous determination of intra- and extracellular oxygen concentrations [partial pressure of O(2) (pO(2))] in bovine lung microvascular endothelial cells (BLMVECs) using electron paramagnetic resonance (EPR) oximetry. The measurements were performed in BLMVEC suspensions of a 20- micro l volume containing 4,000 cells. The extracellular pO(2) was measured using a trityl EPR probe [triarylmethyl (TAM), 10 micro M], a tricarboxylate anion radical, that stays exclusively in the extracellular space. The intracellular oxygen was measured using a pre-internalized particulate spin probe, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-naphthalocyanine (LiNc-BuO). Because there is a wide discrepancy in the reported values of cellular oxygenation by and large due to differences in the methods employed, we utilized the dual EPR probe technique to measure the oxygen gradient that apparently exists across the cell membrane. The intra- and extracellular pO(2) values were 139 +/- 2.5 and 157 +/- 3.6 mm Hg, respectively, for cells exposed to room air. A fairly smaller gradient of oxygen was observed in cells exposed to 7.5% oxygen (pO(2) = 57 mm Hg). In summary, this study confirms the feasibility of simultaneous and accurate measurements of intra- and extracellular pO(2) using LiNc-BuO and TAM EPR oximetry probes.

In vivo imaging of changes in tumor oxygenation during growth and after treatment.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen‐sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen‐sensing probes, which become permanently embedded in the developed tumor. Radiation‐induced fibrosarcoma (RIF‐1) cells, internalized with nanoprobes of lithium octa‐n‐butoxy‐naphthalocyanine (LiNc‐BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30‐Gy X‐radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen‐sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors. Magn Reson Med 57:950–959, 2007.

Myocardial oxygenation and functional recovery in infarct rat hearts transplanted with mesenchymal stem cells

Stem cell therapy for myocardial tissue repair is limited by the poor survival of transplanted cells, possibly because of inadequate supply of oxygen and nutrients. The purpose of this study was to assess the oxygenation level and functional recovery after allogenic transplantation of mesenchymal stem cells (MSC) in a rat model of myocardial infarction (MI). Myocardial oxygen tension (Po(2)) was measured by electron paramagnetic resonance oximetry using an implantable oxygen-sensing spin probe (OxySpin). MSCs incubated with OxySpins showed substantial uptake of the probe without affecting its oxygen sensitivity or calibration. The cells internalized with OxySpins were able to differentiate into osteogenic, adipogenic, cardiomyocyte, and endothelial cell lineages. The labeled cells tested positive for CD44 and CD29 markers and negative for the hematopoietic markers CD14 and CD45. For the in vivo studies, MI was induced in rats by permanently ligating the left anterior descending coronary artery. MSCs with OxySpins were transplanted in the infarct region of hearts. A significant increase in Po(2) was observed in the MSC group compared with the untreated MI group (18.1 +/- 2.6 vs. 13.0 +/- 1.8 mmHg, n = 4, P < 0.05) at 4 wk after transplantation. Echocardiography showed a significant improvement in ejection fraction and fraction shortening, which inversely correlated with the magnitude of fibrosis in the treated hearts. The cell-transplanted hearts also showed an increase in vascular endothelial growth factor level and capillary density in the infarct region. The study established our ability to measure and correlate changes in myocardial tissue oxygenation with cardiac function in infarcted rat hearts treated with MSCs.

Oxygen sensitivity and biocompatibility of an implantable paramagnetic probe for repeated measurements of tissue oxygenation

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO2 from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.

The open molecular framework of paramagnetic lithium octabutoxy-naphthalocyanine: implications for the detection of oxygen and nitric oxide using EPR spectroscopy

The oxygen-induced broadening of the electron paramagnetic resonance (EPR) spectrum of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) is interpreted in terms of its open molecular framework crystal structure. LiNc-BuO was prepared as a microcrystalline powder and its structure analyzed using X-ray powder diffraction techniques. The structure contains strongly coupled dimers of LiNc-BuO molecules, which favors a high degree of spin exchange, and results in a single sharp EPR line. The molecular packing leads to a structure with open channels large enough (10 × 6 A2) for the penetration of small diatomic paramagnetic molecules such as oxygen (O2) and nitric oxide (NO), as well as the larger triatomic species, nitrogen dioxide (NO2). The EPR linewidth of LiNc-BuO is extremely sensitive to the concentration of paramagnetic gases in the pressure range of 0–760 mmHg. The effect of oxygen on LiNc-BuO is reversible without any signs of permanent adsorption or chemical oxidation. The time response of the effect of oxygen is extremely rapid (0.24 s). The paramagnetic gas-sensing properties of LiNc-BuO are attributed to the open molecular framework of the crystal structure. The oxygen-sensing property, combined with the previously established biostability and biocompatibility of this material, should enable precise and accurate measurements of oxygen concentration in biological systems using EPR spectroscopy.

Lipopolysaccharide-induced alterations in oxygen consumption and radical generation in endothelial cells

Oxygen consumption rate (OCR) and generation of superoxide and nitric oxide (NO) in mouse aortic endothelial cells (MAECs) treated with lipopolysaccharide (LPS) were studied. The OCR was determined in cell suspensions at 37 °C by electron paramagnetic resonance (EPR) spectroscopy. LPS significantly altered the OCR in a dose and time-dependent fashion. The OCR was significantly elevated immediately following the treatment of MAECs with LPS (5 and 10 μg/ml) and NADPH (100 μM) whereas the same was depressed 1 h after exposure to similar conditions of incubation. Under similar experimental conditions, superoxide generation was also determined by EPR spectroscopy and cytochrome c reduction assays. A marginal increase in the superoxide production was observed when the cells were treated with LPS and NADPH alone whereas the same was further enhanced significantly when the cells were treated with LPS and NADPH together. The increase in oxygen consumption and superoxide production caused by LPS was inhibited by diphenyleneiodonium (DPI), suggesting the involvement of NAD(P)H oxidase. A significant increase in the NO production by MAECs was noticed 1 h after treatment with LPS and was inhibited by L-NAME, further suggesting the involvement of nitric oxide synthase (NOS). Thus, on a temporal scale, LPS-induced alterations in oxygen consumption by MAECs may be under the control of dual regulation by NAD(P)H oxidase and NOS. (Mol Cell Biochem 278: 119–127, 2005)

Fabrication and physical evaluation of a polymer-encapsulated paramagnetic probe for biomedical oximetry.

Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) is a promising probe for biological electron paramagnetic resonance (EPR) oximetry and is being developed for clinical use. However, clinical applicability of LiNc-BuO may be hindered by potential limitations associated with biocompatibility, biodegradation, and migration of individual crystals in tissue. To overcome these limitations, we have encapsulated LiNc-BuO crystals in polydimethyl siloxane (PDMS), an oxygen-permeable and bioinert polymer, to fabricate conveniently implantable and retrievable oxygen-sensing chips. Encapsulation was performed by a simple cast-molding process, giving appreciable control over size, shape, thickness and spin density of chips. The in vitro oxygen response of the chip was linear, reproducible, and not significantly different from that of unencapsulated crystals. Cast-molding of the structurally-flexible PDMS enabled the fabrication of chips with tailored spin densities, and ensured non-exposure of embedded LiNc-BuO, mitigating potential biocompatibility/toxicological concerns. Our results establish PDMS-encapsulated LiNc-BuO as a promising candidate for further biological evaluation and potential clinical application.

Detection and Imaging of Superoxide in Roots by an Electron Spin Resonance Spin-Probe Method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plants extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probes narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plants root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.