Ke Jian Liu

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, HO* adducts generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.

Non-invasive in vivo characterization of release processes in biodegradable polymers by low-frequency electron paramagnetic resonance spectroscopy.

Using stable free radicals (nitroxides) whose spectra reflect microviscosity and pH, low-frequency electron paramagnetic resonance (EPR) spectroscopy was used to characterize the release pattern of subcutaneous implants of poly(D,L-lactide-co-glycolide) (PLGA) continuously and non-invasively in living mice. No significant changes occurred during the first days after implantation. After about 1 week, the recorded EPR spectra gave direct evidence for the formation of compartments with high mobility and increasing acidity in the delivery system. The contribution of the mobile part of the spectrum increased with time, but no remarkable decay of the overall signal intensity was observed during the second week. The EPR signals decayed rapidly after 3 weeks. The experimental data are consistent with bulk hydrolysis as the dominating mechanism of release and are not consistent with a surface-controlled pattern of degradation. The formation of acidic compartments in the delivery system may have significant effects on drug stability, drug solubility, bioavailability, pharmacokinetics, and ultimately on therapeutic efficiency. In particular, the finding of areas of low pH within the polymer raise the possibility that hydrolysable drugs may undergo degradation in the implant prior to their release. Our results demonstrate that EPR is a valuable tool for characterizing such drug delivery systems in vivo.

Evaluation of DEPMPO as a spin trapping agent in biological systems.

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.

In vivo measurement of oxygen concentration using sonochemically synthesized microspheres.

Proteinaceous microspheres filled with nitroxides dissolved in an organic liquid have been synthesized for the first time using high intensity ultrasound; these were used to measure oxygen concentrations in living biological systems. The microspheres have an average size of 2.5 microns, and the proteinaceous shell is permeable to oxygen. Encapsulation of the nitroxides into the microsphere greatly increased the sensitivity of the electron paramagnetic resonance signal line width to oxygen because of the higher solubility of oxygen in organic solvents. The encapsulation also protected the nitroxide from bioreduction. No decrease in intensity of the electron paramagnetic resonance signal was observed during 70 min after intravenous injection of the microspheres into a mouse. Measurement of the changes in oxygen concentration in vivo by means of restriction of blood flow, anesthesia, and change of oxygen content in the respired gas were made using these microspheres.

In vivo EPR dosimetry of accidental exposures to radiation: experimental results indicating the feasibility of practical use in human subjects

Low frequency electron paramagnetic resonance (EPR) provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth in situ, i.e. without removing the teeth from the potential victim. The potential limiting factors for making such measurements are: (1) whether low frequency EPR is sufficiently sensitive to detect radiation-induced signal in human teeth; (2) whether sufficient sensitivity can be maintained under in vivo conditions. In this manuscript, we summarize results indicating that this approach is feasible. Using 1.2 GHz EPR spectroscopy, we found that the lower limit for these measurements in isolated human teeth is 0.2 Gy or lower. Measurements of radiation-induced EPR signals in the teeth of living rats were achieved with sufficient sensitivity to indicate that, when taking into consideration the larger mass of human teeth, similar measurements in human teeth in situ would provide sensitivity in the dose range for potential accidental exposures. We estimate that the current lower limit for detecting radiation doses in human teeth in situ (in vivo) is 0.5-1.0 Gy; this would be sufficient for determining if a person has been exposed to potentially life threatening doses of ionizing radiation. The limiting factor for sensitivity appears to be background signals rather than signal/noise, and there are feasible means to overcome this problem and further increase sensitivity. The additional instrumental developments required to make an effective in vivo EPR dosimetric spectrometer for the measurements in teeth in human subjects in situ, seem quite achievable.

The pO2 in a Murine Tumor after Irradiation: An In Vivo Electron Paramagnetic Resonance Oximetry Study

Using electron paramagnetic resonance (EPR) oximetry with the oxygen-sensitive paramagnetic material, fusinite, we have measured the partial pressure of oxygen (pO2) in the mouse mammary adenocarcinoma MTG-B. The average pO2 in untreated tumors was low (about 5 mm Hg) and decreased with tumor growth. Magnetic resonance imaging and histological examination were used to localize the position of the fusinite with respect to tumor margins and vascularization. The pO2 was generally higher in the periphery than in the center of the tumors, but there was considerable variation among tumors both during normal growth and after radiation treatment. After a single 20-Gy dose, a characteristic pattern of change in tumor pO2 was observed. In irradiated tumors, there was an initial reduction in pO2 (minimum occurred 6 h postirradiation) which was followed by a transient increase in pO2 to levels higher than the preirradiation pO2 (maximum occurred 48 h postirradiation). This work demonstrates postirradiation changes in pO2 of potential radiobiological significance. Compared to other oxygen assessment techniques, EPR oximetry is very useful because it can assess pO2 in the same region of the tumor over the course of tumor growth and during response to treatment. Thus EPR could be used to identify potentially radioresistant tumors as well as to identify tumors with slow reoxygenation.

Effect of Anesthesia on Cerebral Tissue Oxygen and Cardiopulmonary Parameters in Rats

General anesthesia is known to alter cardiopulmonary and hematological parameters which affect tissue oxygenation. However, the effect of various types of anesthetics on the relationship between brain tissue pO2 and these physiologic parameters is still largely unknown.

Comparisons of Measurements of pO2 in Tissue In Vivo by EPR Oximetry and Micro-Electrodes

Polarographic micro-electrode measurements are very useful for measuring pO2 in vivo, especially for measurements of the variation of pO2 within a tumor (1,2,8). This method has several advantages, including: it is the only direct method currently in extended use in the clinical setting; it can provide data on microscopic heterogeneity; and it is fairly widely available. While the micro-electrode method has become a type of “gold standard” for measurement of pO2 in tissues, it has some limitations and disadvantages: it can be technically difficult; it has limited resolution at the very low levels of pO2 that are important for many clinically relevant processes; and it can perturb the tissues significantly, especially when used in repeated studies to monitor pO2 in tissues over time. Repeated measurements are especially desirable to follow the effect on tissue pO2 after treatment with some drugs (e.g. anti-cancer drugs and anesthetics) and radiation, the effects of acute and chronic ischemia, and changes in respiratory factors. Electron paramagnetic resonance (EPR) oximetry appears to offer some complimentary advantages for such studies: it can monitor pO2 continuously and/or repeatedly at the exactly the same localized area in tissue in vivo without the need for anesthesia; it can resolve small differences in pO2 even at the very low levels that occur pathophysiologically; and it can be used in a variety of settings.

Measurements of pO2 in Vivo, Including Human Subjects, by Electron Paramagnetic Resonance

The purpose of this paper is to provide an illustrative description of the current state of development of the use of electron paramagnetic resonance (EPR, or completely equivalently, electron spin resonance or ESR) to measure the partial pressure of oxygen (pO2) in tissues in vivo under physiological conditions. This summary is based on published and unpublished results from our laboratory (1–7) and does not attempt to describe the results of other laboratories which also are working along related lines (8–10). The pertinent features of our technique are illustrated. We also consider the current limitations of the technique and likely developments in the near future. Our evaluation is that: this technique now is suitable for immediate use in small animals; within a short period of time instruments will be available facilitating its use in larger animals; and preliminary studies are imminent in human subjects (7).

The Measurement of Temperature With Electron Paramagnetic Resonance Spectroscopy

An electron paramagnetic resonance (EPR) technique, potentially suitable for in vivo temperature measurements, has been developed based on the temperature response of nitroxide stable free radicals. The response has been substantially enhanced by encapsulating the nitroxide in a medium of a fatty acid mixture inside a proteinaceous microsphere. The mixture underwent a phase transition in the temperature range required by the application. The phase change dramatically altered the shape of the EPR spectrum, providing a highly temperature sensitive signal. Using the nitroxide dissolved in a cholesterol and a long-chain fatty acid ester, we developed a mixture which provides a peakheight ratio change from 3.32 to 2.11, with a standard deviation of 0.04, for a temperature change typical in biological and medical applications, from 38 to 48 degrees C. This translated to an average temperature resolution of 0.2 degree C for our experimental system. The average diameter of the nitroxide mixture-filled microspheres was approximately 2 microns. Therefore, they are compatible with in vivo studies where the microspheres could be injected into the microvasculature having a minimum vessel diameter of the order of 8 microns. This temperature measuring method has various potential clinical applications, especially in monitoring and optimizing the treatment of cancer with hyperthermia. However, several problems regarding temperature and spatial resolution need to be resolved before this technique can be successfully used to monitor temperatures in vivo.