Optical properties of human prostate at 732 nm measured in mediated photodynamic therapy

Characterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [mu(a)], transport scattering [mu(s)'] and effective attenuation [mu(eff)] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)-mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5-5 cm) from a point source. mu(a) and mu(s)' varied between 0.07 and 1.62 cm(-1) (mean 0.37 +/- 0.24 cm(-1)) and 1.1 and 44 cm(-1) (mean 14 +/- 11 cm(-1)), respectively. mu(a) was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. mu(eff) varied between 0.91 and 6.7 cm(-1) (mean 2.9 +/- 0.7 cm(-1)), corresponding to an optical penetration depth (delta = 1/micro(eff)) of 0.1-1.1 cm (mean 0.4 +/- 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of mu(s)' in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 +/- 1.1 cm(-2), excluding situations when bleeding occurs. The total number of measurements was N = 121 for all mean quantities listed above. This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.     

Determination of the distribution of light, optical properties, drug concentration, and tissue oxygenation in-vivo in human prostate during motexafin lutetium-mediated photodynamic therapy

It is desirable to quantify the distribution of the light fluence rate, the optical properties, the drug concentration, and the tissue oxygenation for photodynamic therapy (PDT) of prostate cancer. We have developed an integrated system to determine these quantities before and after PDT treatment using motorized probes. The optical properties (absorption (micro(a)), transport scattering (micro(s'), and effective attenuation (micro(eff)) coefficients) of cancerous human prostate were measured in-vivo using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu) mediated PDT at different locations along each catheter. The light fluence rate distribution was also measured along the catheters during PDT. Diffuse absorption spectroscopy measurement using a white light source allows extrapolation of the distribution of oxygen saturation StO2, total blood volume ([Hb]t), and MLu concentration. The distribution of drug concentration was also studied using fluorescence from a single optical fiber, and was found to be in good agreement with the values determined by absorption spectroscopy. This study shows significant inter- and intra-prostatic variations in the tissue optical properties and MLu drug distribution, suggesting that a real-time dosimetry measurement and feedback system for monitoring these values during treatment should be considered in future PDT studies.     

Interstitial fluorescence spectroscopy in the human prostate during motexafin lutetium-mediated photodynamic therapy

The in vivo fluorescence emission from human prostates was measured before and after motexafin lutetium (MLu)-mediated photodynamic therapy (PDT). A single side-firing optical fiber was used for both the delivery of 465 nm light-emitting diode excitation light and the collection of emitted fluorescence. It was placed interstitially within the prostate via a closed transparent plastic catheter. Fitting of the collected fluorescence emission spectra using the known fluorescence spectrum of 1 mg/kg MLu in an intralipid phantom yields a quantitative measure of the local MLu concentration. We found that an additional correction factor is needed to account for the reduction of the MLu fluorescence intensity measured in vivo due to strong optical absorption in the prostate. We have adopted an empirical correction formula given by C = (3.1 cm(-1)/micro's) exp (microeff x 0.97 cm), which ranges from approximately 3 to 16, with a mean of 9.3 +/-4.8. Using a computer-controlled step motor to move the probe incrementally along parallel tracks within the prostate we can determine one-dimensional profiles of the MLu concentration. The absolute MLu concentration and the shape of its distribution are confirmed by ex vivo assay and by diffuse absorption measurements, respectively. We find significant heterogeneity in photosensitizer concentration within and among five patients. These variations occur over large enough spatial scales compared with the sampling volume of the fluorescence emission that mapping the distribution in three dimensions is possible.     

Dose and timing of the first light fraction in two-fold illumination schemes for topical ALA-mediated photodynamic therapy of hairless mouse skin

A fractionated illumination scheme in which a cumulative fluence of 100 J cm(-2) is delivered in two equal light fractions separated by a dark interval of 2 h has been shown to considerably increase the efficacy of 5-aminolevulinic acid (ALA)-photodynamic therapy (PDT). The efficacy of such a scheme is further increased if the fluence of the first light fraction is reduced to 5 J cm(-2). We have investigated the relationship between the PDT response and the kinetics of protoporphyrin IX (PpIX) fluorescence in the SKH1 HR hairless mouse for first fraction fluences below 5 J cm(-2) delivered 4 h after the application of ALA and 10 J cm(-2) delivered 2 h after the application of ALA. Illumination is performed using 514 nm at a fluence rate of 50 mW cm(-2). Reducing the fluence of the first fraction to 2.5 J cm(-2) does not result in significantly different visual skin damage. The PDT response, however, is significantly reduced if the fluence is lowered to 1 J cm(-2), but this illumination scheme (1 + 99 J cm(-2)) remains significantly more effective than a single illumination of 100 J cm(-2). A first light fraction of 10 J cm(-2) can be delivered 2 h earlier, 2 h after the application of ALA, without significant reduction in the PDT response compared with 5 + 95 J cm(-2) delivered 4 and 6 h after the application of ALA. The kinetics of PpIX fluorescence are consistent with those reported previously by us and do not explain the significant increase in PDT response with a two-fold illumination scheme. Histological sections of the illuminated volume showed a trend toward increasing extent and depth of necrosis for the two-fold illumination scheme in which the first light fraction is 5 J cm(-2), compared with a single illumination scheme.     

The Effect of Fluence Rate on Tumor and Normal Tissue Responses to Photodynamic Therapy

Photodynamic therapy (PDT), carried out at low fluence rates, may enhance tumor response as well as affect treatment selectivity. We have studied the effects of fluence rate on the response of the murine radiation-induced fibrosarcoma (RIF) to PDT using Photofrin® (5 mg/kg). Tumor response was tested over a large range of fluence rates (10-200 mW/cm²) and fluences (25-378 J/ cm²). Low fluence rates were more efficient; -60 J/cm2 at 10 mW/cm² was needed to achieve the same tumor growth delay as -100 J/cm² at 150 mW/cm² and -150 J/cm2 at 200 mW/cm². Despite this increased efficiency, lower fluence rates still required longer treatment times for equivalent anti-tumor effects: 95 min for 57 J/cm² at 10 mW/cm²versus 11 min for 100 J/cm² at 150 mW/cm². Effects of fluence rate on the PDT toxicity to normal tissue were examined through the response of the murine (C311) foot to Photofrin® PDT. Treatment with conditions that produced equivalent tumor responses, i.e. 57 J/cm² at 10 mW/cm² and 100 J/cm² at 150 mW/cm², resulted in a more severe foot response at the higher fluence rate (median peak response: 0.9 at 10 mW/cm², 1.5 at 150 mW/cm²) with more time required for tissue to return to normal (8 days at 10 mW/cm², at least 30 days at 150 mW/cm²). However, when feet were treated with an equal fluence of 100 J/cm2 at various fluence rates, longer healing times accompanied the lower fluence rate treatments. Overall, this paper demonstrates that lower PDT fluence rates are associated with increased efficiency of tumor response. If this increased efficiency is accounted for by lowering treatment fluence, lower fluence rates also may result in a more favorable normal tissue response to treatment.     

Effects of combined photodynamic therapy and ionizing radiation on human glioma spheroids

The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm(-2) and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm(-2)) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.     

Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent

The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation-induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin (5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 15% tumor cures, a response not significantly altered by administration of 20 mg/kg L-NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L-NNA either pre- or post-PDT resulted in approximately 30% and approximately 10% tumor cures, respectively. The L-NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L-NNA before PDT further potentiated this effect. The L-NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut-down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L-NNA reversed this response, converting it to complete and lasting vascular shut-down identical to that achieved with low fluence rate PDT. This effect was somewhat L-NNA dose dependent but was still marked at a dose of 1 mg/kg. It occurred whether L-NNA was given before or after PDT. The L-NNA did not alter the long-term vascular response of skin to low fluence rate PDT. The ability of L-NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L-NNA treatment.     

A Comparison of Dose Metrics to Predict Local Tumor Control for Photofrin‐mediated Photodynamic Therapy

This preclinical study examines light fluence, photodynamic therapy (PDT) dose and “apparent reacted singlet oxygen,” [¹O₂]_rx, to predict local control rate (LCR) for Photofrin‐mediated PDT of radiation‐induced fibrosarcoma (RIF) tumors. Mice bearing RIF tumors were treated with in‐air fluences (50–250 J cm^?2) and in‐air fluence rates (50–150 mW cm^?2) at Photofrin dosages of 5 and 15 mg kg^?1 and a drug‐light interval of 24 h using a 630‐nm, 1‐cm‐diameter collimated laser. A macroscopic model was used to calculate [¹O₂]_rx and PDT dose based on in vivo explicit dosimetry of the drug concentration, light fluence and tissue optical properties. PDT dose and [¹O₂]_rx were defined as a temporal integral of drug concentration and fluence rate, and singlet oxygen concentration consumed divided by the singlet oxygen lifetime, respectively. LCR was stratified for different dose metrics for 74 mice (66 + 8 control). Complete tumor control at 14 days was observed for [¹O₂]_rx ≥ 1.1 mm or PDT dose ≥1200 μm J cm^?2 but cannot be predicted with fluence alone. LCR increases with increasing [¹O₂]_rx and PDT dose but is not well correlated with fluence. Comparing dosimetric quantities, [¹O₂]_rx outperformed both PDT dose and fluence in predicting tumor response and correlating with LCR.     

Topical photodynamic therapy at low fluence rates--theory and practice

Photodynamic Therapy (PDT), with topically applied 5-aminolaevulinic acid as the photosensitiser, is an effective treatment for various malignant and pre-malignant skin conditions. Several studies have shown the importance of fluence rate as well as fluence in the efficacy of PDT. We propose a measure of PDT efficacy, Photodynamic Damage Dose (PDD), which uses the product of instantaneous fluence rates, photosensitiser concentrations and oxygen concentrations in its calculation. We derive a qualitative numerical model of PDT and verify it by demonstrating an inverse fluence rate effect, increased efficacy of fractionated PDT, PDT induced hypoxia, and the dependence of photobleaching on fluence rate under certain circumstances. We recommend that fluence, fluence rate and any fractionation regime used should be detailed when reporting a trial as altering any of these has significant effects on PDT efficacy. The model predicts that low fluence rate irradiations should be as effective as high fluence rate irradiations if carried out over the same length of time. To test this we build a light emitting diode-based lamp (fluence rate of 7 mW cm(-2) at 635 nm) and used it to treat 32 superficial basal cell carcinomas on 22 patients (30 min treatment time, fluence 12.6 J cm(-2)). The complete response rate at one year was 84%, which is comparable to that achieved using higher fluence rate sources for similar treatment times. We conclude that this robust, inexpensive light source is effective for topical PDT.     

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.     

Intraperitoneal Photodynamic Therapy: Comparison of Red and Green Light Distribution and Toxicity

Abstract— The aim of this study was to compare red (652 nm) and green (514 nm) light for photodynamic therapy (PDT) of the peritoneal cavity with emphasis on light distribution and toxicity. Red-light PDT was limited by intestinal toxicity and it was hypothesized that less penetrating green light would allow higher light doses to be used in the peritoneal cavity. Female non-tumor-bearing rats were photosensitized with mTHPC (meta-tetrahydroxyphenylchlorin, Foscan®) intravenously or intraperitoneally and the peritoneum was illuminated using a minimally invasive technique. For both red and green light, the time of illumination was varied to give the required dose. Light fluence rate was measured in situ at multiple sites within the abdominal cavity. The toxicity experiments were carried out with a total of 160 J incident red or 640 J incident green light and a drug dose of 0.15 mg/kg Foscan® For red light a mean fluence rate of 55.2 38.5 mW cm ₂ was measured, with a peak fluence rate of 128 mW cm ₂ on the intestines. For green light the mean and peak fluence rates were 8.2 9.0 (i.e. including zero fluence rate measurements) and 28 mW cm ₂, respectively. Intestines were most vulnerable to red light illumination. The intravenous injection route resulted in increased toxicity for red light, but for green light there were no major differences between intravenous and intraperitoneal routes. The 4 h interval between drug and illumination resulted in very little toxicity for both wavelengths. We conclude that for intraperitoneal PDT green light allows higher light doses than red light, but the light distribution over the peritoneum is much less favorable and may not be suitable for whole peritoneal illumination using a minimal-access technique.     

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin-PDT of radiation-induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25-225 mW cm(-2) and 2.5-10 mg kg(-1), respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mWx2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mWx2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mWx2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen-conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.     

Cyanide-limited complexation of molybdenum(III): synthesis of octahedral [Mo(CN)(6)](3-) and cyano-bridged [Mo(2)(CN)(11)](5-)

Octahedral coordination of molybdenum(III) is achieved by limiting the amount of cyanide available upon complex formation. Reaction of Mo(CF(3)SO(3))(3) with LiCN in DMF affords Li(3)[Mo(CN)(6)] x 6DMF (1), featuring the previously unknown octahedral complex [Mo(CN)(6)](3-). The complex exhibits a room-temperature moment of mu(eff) = 3.80 mu(B), and assignment of its absorption bands leads to the ligand field parameters Delta(o) = 24800 cm(-1) and B = 247 cm(-1). Further restricting the available cyanide in a reaction between Mo(CF(3)SO(3))(3) and (Et(4)N)CN in DMF, followed by recrystallization from DMF/MeOH, yields (Et(4)N)(5)[Mo(2)(CN)(11)] x 2DMF x 2MeOH (2). The dinuclear [Mo(2)(CN)(11)](5-) complex featured therein contains two octahedrally coordinated Mo(III) centers spanned by a bridging cyanide ligand. A fit to the magnetic susceptibility data for 2, gives J = -113 cm(-1) and g = 2.33, representing the strongest antiferromagnetic coupling yet observed through a cyanide bridge. Efforts to incorporate these new complexes in magnetic Prussian blue-type solids are ongoing.     

Real-time in situ monitoring of human prostate photodynamic therapy with diffuse light

Photodynamic therapy (PDT) requires oxygen to cause cellular and vascular tumor damage. Tissue oxygen concentration, in turn, is influenced by blood flow and blood oxygenation. Real-time clinical measurement of these hemodynamic quantities, however, is rare. This paper reports the development and application of a probe, combining diffuse reflectance spectroscopy (DRS) for measurement of tumor blood oxygenation and diffuse correlation spectroscopy (DCS) for measurement of tumor blood flow. The instrument was adapted for clinical use during interstitial prostate PDT. Three patients with locally recurrent prostate cancer received 2 mg/ kg motexafin lutetium (MLu) 3 h before illumination and a total light dose of 100 J/cm(2) at 150 mW/cm. Prostrate blood oxygen saturation (StO2) decreased only slightly (approximately 3%) after treatment. On the other hand, prostate blood flow and total hemoglobin concentration over the course of PDT decreased by 50% and 15%, respectively, suggesting MLu-mediated PDT has an anti-vascular effect. While it is certainly impossible to draw definite conclusions from measurements of only three patients, the observed differences in tumor blood flow and blood oxygenation responses during PDT can, in principle, be used to choose among tissue oxygen consumption models and therefore emphasize the potential clinical value for simultaneous monitoring of both parameters.     

Fluence Rate or Cumulative Dose? Vulnerability of Larval Northern Pike (Esox lucius) to Ultraviolet Radiation

Newly hatched larvae of northern pike were exposed in the laboratory to four fluence rates of ultraviolet radiation (UVR; 290-400 nm) over three different time periods, resulting in total doses ranging from 3.0 +/- 0.2 to 63.0 +/- 4.4 kJ.m(-2). Mortality and behavior of the larvae were followed for 8-12 days, and growth measured at the end of the experiment. Also, the principle of reciprocity-that the UVR-induced mortality depends on the cumulative dose, independent of fluence rate-was tested. Fluence rates higher than 1480 +/- 150 mW.m(-2) caused mortality and growth retardation. The highest fluence rate (3040 +/- 210 mW.m(-2)) caused 100% mortality in 5 days. All fluence rates caused behavioral disorders, which led to death at fluence rates higher than 1480 mW.m(-2). Reciprocity failure occurred with the lowest and highest dose (550 +/- 45 and 3040 +/- 210 mW.m(-2), respectively). The results show that fluence rate is of primary importance when assessing the UVR-related risk.     

Magneto-optics of Ferritin

Measurements of Rayleigh light scattering, nonlinear light scattering in DC magnetic fields, and the Cotton-Mouton effect were carried out for 15 mM NaCl and water solutions of ferritin at room temperature. The spherical geometry of the molecule implies that it is optically isotropic. Such a macromolecule should not manifest magnetic anisotropy; however, in solution it shows induced magnetic birefringence (Cotton-Mouton effect) and changes in the intensity of the scattered light components. The analysis of the obtained results indicates the deformation of linear optical polarizability induced in the ferritin by a magnetic field as the main source of the magneto-optical phenomena observed. Light scattering and the CM effects theoretically depend on the linear magneto-optical polarizability, chi, and the nonlinear magneto-optical polarizability, eta. Using the theory describing the phenomena as well as the experimental data, the values of the anisotropy of linear magneto-optical polarizability components, chi(parallel) - chi(perpendicular) = -(1.3 +/- 0.7) x 10(-22) [cm3] (in SI units chi(parallel) - chi(perpendicular) = -(2.0 +/- 1.2) x 10(-33) [m3]), the linear optical polarizability, alpha = (alpha(parallel) + 2alpha(perpendicular))/3 = (3.9 +/- 1.0) x 10(-20) [cm3] (in SI units alpha = (3.52 +/- 0.09)x10(-4) [Cm2 V(-1)]), and its anisotropy, kappa(alpha) = (alpha(parallel) - alpha(perpendicular))/3alpha = -(0.06+/-0.03), nonlinear magneto-optical polarizability, eta = (eta(parallel) + 2eta(perpendicular))/3 = -(4.7 +/- 0.9) x 10(-30) [cm3 Oe(-2)] (in SI units eta = -(6.7 +/- 1.3) x 10(-18) [Cm4 V(-1) A(-2)]) and its anisotropy, kappa(eta) = (eta[parallel) - eta(perpendicular))/3eta = -(0.15 +/- 0.10), were deduced. Here alpha(parallel), eta(parallel), alpha(perpendicular), eta(perpendicular) are the optical and magneto-optical polarizability components along the parallel and the perpendicular axes of the axially symmetric molecule, respectively.     

An unusual (10,3)-a racemic twofold interpenetrating network assembled from isolable tris(cyclopentadienyl)manganate and cesocene building blocks

The syntheses and X-ray crystal structures of [([18]crown-6)2Cs](+)-[Cp3Mn]- (1), [([18]crown-6)2Cs](+)-[Cp'3Mn]- (2), [CsCp'] (3), [(CsCp')2-([18]crown-6)] (4), and Cs[MnCp3] (5), and the synthesis of Cs[MnCp'3] (6) are reported (Cp' = C5H4Me). The anions [Cp3Mn]- (1-) and [Cp'3Mn]- (2-) are characterized by eta 2 coordination of all three Cp or Cp' rings. Measurements of the magnetic susceptibilities chi M resulted in values of mu eff = 6.20 microB (300 K), mu eff = 6.33 microB (301 K), and mu eff = 5.83 microB (300 K) for 1, 2, and 5, respectively, which are indicative of high-spin d5-Mn2+ centers. Density functional calculations illustrate that the coordination mode of 1- is characteristic for its sextet electronic ground state. Compound 3 forms infinite chains of cesocene-type sandwiches in the solid state, which are broken up into small subunits by the addition of crown ether to form 4. Compound 5 is a rare example of a (10,3)-a racemic interpenetrating network that crystallizes in the orthorhombic space group Pbca.     

Monitoring ALA-induced PpIX photodynamic therapy in the rat esophagus using fluorescence and reflectance spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence of 54 J cm(-2). Forty-eight hours after PDT the esophagus was excised and histologically examined for PDT-induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two-phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two-phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.     

Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P<0.05) in normal mouse skin than in tumours. In addition, for a given fluence, more PpIX was photobleached at the lower fluence rate (10 mW/cm2) than at the higher fluence rate (40 mW/cm2) in normal skin (P<0.001) as well as in tumours (P<0.05) after exposure to 24 J/cm2 of light. In conclusion, the highest tumour to normal skin PpIX ratio was observed 3 h after application of 8% ALA-Me, suggesting that light exposure should be performed at this time in order to achieve an optimal PDT effect in this tumour model.     

Photodynamic actinometry using microspheres: concept, development and responsivity

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.