1O2 Quantum Yield Research Articles

Hypericin-loaded lipid nanocapsules for photodynamic cancer therapy in vitro

Hypericin (Hy), a naturally occurring photosensitizer (PS), is extracted from Hypericum perforatum plants, commonly known as St. John's wort. The discovery of the in vitro and in vivo photodynamic activities of hypericin as a photosensitizer generated great interest, mainly to induce a very potent antitumoral effect. However, this compound belongs to the family of naphthodianthrones which are known to be poorly soluble in physiological solutions and produce non-fluorescent aggregates (A. Wirz et al., Pharmazie, 2002, 57, 543; A. Kubin et al., Pharmazie, 2008, 63, 263). These phenomena can reduce its efficiency as a photosensitizer for the clinical application. In the present contribution, we have prepared, characterized, and studied the photochemical properties of Hy-loaded lipid nanocapsule (LNC) formulations. The amount of singlet oxygen ((1)O2) generated was measured by the use of p-nitroso-dimethylaniline (RNO) as a selective scavenger under visible light irradiation. Our results showed that Hy-loaded LNCs suppressed aggregation of Hy in aqueous media, increased its apparent solubility, and enhanced the production of singlet oxygen in comparison with free drug. Indeed, encapsulation of Hy in LNCs led to an increase of (1)O2 quantum yield to 0.29-0.44, as compared to 0.02 reported for free Hy in water. Additionally, we studied the photodynamic activity of Hy-loaded LNCs on human cervical carcinoma (HeLa) and Human Embryonic Kidney (HEK) cells. The cell viability decreased radically to 10-20% at 1 μM, reflecting Hy-loaded LNC25 phototoxicity.

Tetra-2,3-pyrazinoporphyrazines with Externally Appended Pyridine Rings. 12. New Heteropentanuclear Complexes Carrying Four Exocyclic Cis-platin-like Functionalities as Potential Bimodal (PDT/Cis-platin) Anticancer Agents

Heteropentanuclear porphyrazines having the formula [(PtCl2)4LM] where L = tetrakis-2,3-[5,6-di(2-pyridyl)pyrazino]porphyrazinato dianion and M = Zn(II), Mg(II)(H2O), Pd(II), Cu(II) or Co(II) were characterized by elemental analyses, IR-UV-visible spectroscopy and electrochemistry and the data compared to new and previously published results for the corresponding homopentanuclear compound [(PtCl2)4LPt]. This latter species has four external N2(py)PtCl2 coordination sites which closely resemble cis-platin, (NH3)2PtCl2, the potent chemotherapeutic anticancer drug, and is able to act as a photosensitizer for the generation of (1)O2, the cytotoxic agent in photodynamic therapy (PDT). UV-visible spectra and half wave potentials for reduction of [(PtCl2)4LM], [(PtCl2)4LPt], the parallel series of mononuclear [LM] compounds and the pentanuclear [(PdCl2)4LM] compounds were examined in the nonaqueous solvents dimethyl sulfoxide, pyridine, and dimethylformamide. The complete set of available data indicate that external coordination of the PtCl2 and PdCl2 units significantly increases the level of the electron-deficiency of the entire molecular framework despite the fact that these groups are far away from the central porphyrazine π-ring system and have coordination sites nearly orthogonal to the plane of the macrocycle. The pentanuclear species [(M'Cl2)4LM] (M' = Pt(II), Pd(II)) undergo multiple one-electron transfers and exhibit an easier reducibility as compared to related electrode reactions of the parent compounds [LM] having the same central metal. Aggregation phenomena and reducibility of the porphyrazines to their monoanionic form (prevalently in DMF) are observed for some of the examined compounds and were analyzed and accurately taken into account. Quantum yields of (1)O2 (ΦΔ), of interest in PDT, were measured for [(PtCl2)4LM] with M = Zn(II), Mg(II)(H2O), or Pd(II) and the related macrocycles [(PdCl2)4LM] and [LM] in dimethylformamide (DMF) and/or DMF preacidified with HCl (DMF/HCl, [HCl]: 1-2 × 10(-4) M). Excellent ΦΔ values (0.5-0.6) which qualify the compounds as potent photosensitizers in PDT were obtained for the pentanuclear species having Zn(II) or Pd(II) as central metal ions. The [(PtCl2)4LZn] and [(PtCl2)4LPd] complexes are of special interest as potential bimodal anticancer agents because of the incorporated four cis-platin-like functionalities.

Quantum Yield and Mechanism of Singlet Oxygen Generation via UV Photoexcitation of O2–O2 and N2–O2 Encounter Complexes

The mechanism and spectral dependence of the quantum yield of singlet oxygen O(2)(a (1)Δ(g)) photogenerated by UV radiation in gaseous oxygen at elevated pressure (32-130 bar) have been experimentally investigated within the 238-285 nm spectral region overlapping the range of the Wulf bands in the absorption spectrum of oxygen. The dominant channel of singlet oxygen generation with measured quantum yield up to about 2 is attributed to the one-quantum absorption by the encounter complexes O(2)-O(2). This absorption gives rise to oxygen in the Herzberg III state O(2)(A' (3)Δ(u)), which is assumed to be responsible for singlet oxygen production in the relaxation process O(2)(A' (3)Δ(u), υ) + O(2)(X (3)Σ(g)(-)) → O(2)({a (1)Δ(g)}, {b (1)Σ(g)(+)}) + O(2)({a (1)Δ(g), υ = 0}, {b (1)Σ(g)(+), υ = 0}) with further collisional relaxation of b to a state. This mechanism is deduced from the analysis of the avoiding crossing locations on the potential energy surface of colliding O(2)-O(2) pair. The observed drop of the O(2)(a (1)Δ(g)) yield near spectral threshold for O(2) dissociation is explained by the competition between above relaxation and reaction giving rise to O(3) + O (O + O + O(2)) supposed in literature. The quantum yield of O(2)(a (1)Δ(g)) formation from encounter complex N(2)-O(2) measured at λ = 266 nm was found to be the same as that for O(2)-O(2).

Photogeneration of singlet molecular oxygen (1O2, 1Δ g ) using monomeric and aggregated forms of copper tetra-4-(morpholine-4-yl)-tetra-5-(2-naphthoxy)phthalocyanine in organic solvents

Luminescence and chemical acceptors methods were used to study the photosensitized formation of singlet molecular oxygen (1O2, 1Δ g ) by monomer and aggregated forms of copper tetra-4-(morpholine-4-yl)-tetra-5-(2-naphthoxy)phthalocyanine in benzene, benzene-d 6, acetone, DMF, and pyridine. The values of 1O2 quantum yield were determined. The effectiveness of the 1O2 photoproduction was shown to be reduced due mainly to the aggregation of the copper phthalocyanine molecules.

Acetylene bridged porphyrin–monophthalocyaninato ytterbium(iii) hybrids with strong two-photon absorption and high singlet oxygen quantum yield

Several acetylene bridged porphyrin-monophthalocyaninato ytterbium(III) hybrids, PZn-PcYb, PH(2)-PcYb and PPd-PcYb, have been prepared and characterized by (1)H and (31)P NMR, mass spectrometry, and UV-vis spectroscopy. Their photophysical and photochemical properties, especially the relative singlet oxygen ((1)O(2)) quantum yields and the two-photon absorption cross-section (σ(2)), were investigated. These three newly synthesized compounds exhibited very large σ(2) values and substantial (1)O(2) quantum yields upon photo-excitation, making them potential candidates as one- and two-photon photodynamic therapeutic agents.

The synthesis and 1O2 photosensitization of halogenated asymmetric aniline-based squaraines

The halogenated (brominated or iodinated) squaraines have promising potential in the application of photodynamic therapy (PDT). Though aniline-based squaraines are the most classical squaraine dyes, no halogenated derivatives with the halogen atoms directly incorporated in the conjugation skeleton of the squaraine chromophore have been reported so far. In this work, a stepwise synthetic approach, i.e. by way of the halogenated semi-squaraines, was applied successfully to prepare halogenated asymmetric aniline-based squaraines. Such a structure makes iodine atoms be able to exert significant heavy atom effect on the intersystem crossing (ISC) efficiency of the squaraine dye. As a result, the iodinated asymmetric squaraine (SQ–OH–I) exhibits a singlet oxygen (1O2) quantum yield as high as 0.54. In contrast, the 1O2 quantum yield of the non-halogenated squaraine analogue (SQ–OH) is only 0.02.

Ru(bpy)3−n(dpb)n]2+: Unusual Photophysical Property and Efficient DNA Photocleavage Activity

The sequential replacement of a bpy ligand (bpy = 2,2'-bipyridine) by a dpb ligand (dpb = 2,3-bis(2-pyridyl) benzoquinoxaline) in the series [Ru(bpy)(3-n)(dpb)(n)](2+) (n = 1-3) leads to a remarkable increase of the excited state lifetime, the (1)O(2) quantum yield, and the binding affinity toward dsDNA, rendering both [Ru(bpy)(dpb)(2)](2+) and [Ru(dpb)(3)](2+) efficient DNA photocleavage activities upon red light irradiation (>or=600 nm).

A heterogeneous optimization algorithm for reacted singlet oxygen for interstitial PDT.

Singlet oxygen (1O2) is the major cytotoxic agent for type II photodynamic therapy (PDT). The production of 1O2 involves the complex reactions among light, oxygen molecule, and photosensitizer. From universal macroscopic kinetic equations which describe the photochemical processes of PDT, the reacted 1O2 concentration, [1O2]rx, with cell target can be expressed in a form related to time integration of the product of 1O2 quantum yield and the PDT dose rate. The object of this study is to develop optimization procedures that account for the optical heterogeneity of the patient prostate, the tissue photosensitizer concentrations, and tissue oxygenation, thereby enable delivery of uniform reacted singlet oxygen to the gland. We use the heterogeneous optical properties measured for a patient prostate to calculate a light fluence kernel. Several methods are used to optimize the positions and intensities of CDFs. The Cimmino feasibility algorithm, which is fast, linear, and always converges reliably, is applied as a search tool to optimize the weights of the light sources at each step of the iterative selection. Maximum and minimum dose limits chosen for sample points in the prostate constrain the solution for the intensities of the linear light sources. The study shows that optimization of individual light source positions and intensities is feasible for the heterogeneous prostate during PDT. To study how different photosensitizer distributions as well as tissue oxygenation in the prostate affect optimization, comparisons of light fluence rate were made with measured distribution of photosensitizer in prostate under different tissue oxygenation conditions.

The Excited Triplet State Properties of Titanyl Phthalocyanine and its Sulfonated Derivatives

Titanyl phthalocyanine (TiOPc) is a well-known, excellent photoconductive material for laser printers and photocopying machines. Its organic derivatives have recently been shown to be excellent photosensitizers for singlet oxygen [O2(1Δg)] production. The excited triplet state properties of TiOPc, in homogeneous DMSO solution, were measured in this study for the first time by nanosecond laser flash photolysis. The data enabled comparisons to be drawn with TiOPcS4 and zinc phthalocyanine (ZnPc), ultimately providing a better understanding of the reported observations. Absorption, fluorescence, and O2(1Δg) sensitization were also studied. TiOPcS4 in DMSO shows remarkably different fluorescence properties from that reported in aqueous solution: both the fluorescence quantum yield (Φf = 0.068) and the fluorescence lifetime (τf = 3.71 ns) were much larger than that reported for aqueous solutions (0.012 and 0.09 ns, respectively). The photosensitizing properties of TiOPcS4 in DMSO are also so significantly better than that in aqueous solution, i.e. triplet lifetime (τT) of 252 μs, triplet quantum yield (ΦT) of 0.42, and the quantum yield of O2(1Δg) (ΦΔ) of 0.49; compare with values of 60 μs, 0.32, 0.13 reported in aqueous solution. TiOPc, however, shows comparable photophysical properties to that of ZnPc, a well-recognized photosensitizer. These results suggest that TiOPc and its derivatives are not only good photoconductors but also good photosensitizers of O2(1Δg), which may find application in photodynamic therapies for treatment of cancer.

Evolution of the Triplet Excited State in PtII Perylenediimides

Here, we present the ultrafast dynamics of a series of metal complexes developed to permit access to the perylenediimide (PDI) triplet manifold that preserves the desirable colorfastness and visible light-absorption properties associated with these dyes. To this end, three Pt(II) complexes each bearing two PDI moieties tethered to the metal center through acetylide linkages emanating from one of the PDI bay positions have been thoroughly examined by static spectroscopic methods, electrochemistry, laser flash photolysis, and ultrafast transient absorption spectrometry. Upon ligation to the Pt(II) center, the bright singlet-state fluorescence (Phi = 0.91, tau = 4.53 ns) of the free PDI-CCH chromophore is quantitatively quenched, and no long wavelength photoluminescence is observed from any of the Pt(II)-PDI complexes in deaerated solutions. Ultrafast transient measurements reveal that upon ligation of PDI-CCH to the Pt(II) center, picosecond intersystem crossing (tau = 2-4 ps) from the (1)PDI excited state is followed by vibrational cooling (tau = 12-19 ps) of the hot (3)PDI excited state, whereas only singlet-state dynamics, including stimulated emission, were observed in the "free" PDI-CCH moiety. In each of the Pt-PDI chromophores, quantitatively similar transient absorption difference spectra were obtained; the only distinguishing characteristic is in their single-exponential lifetimes (tau = 246 ns, 1.0 mus, and 710 ns). These long-lived (3)PDI excited states are clearly poised for bimolecular electron and energy transfer schemes. In the present case, the latter is demonstrated through bimolecular sensitization of singlet oxygen phosphorescence at approximately 1270 nm in aerated dichloromethane solutions, producing reasonable (1)O(2) quantum yields (Phi(Delta) = 0.40-0.55) across this series of molecules.

Optimization of physiological parameter for macroscopic modeling of reacted singlet oxygen concentration in an in-vivo model.

Singlet oxygen (1O2) is generally believed to be the major cytotoxic agent during photodynamic therapy (PDT), and the reaction between 1O2 and tumor cells define the treatment efficacy. From a complete set of the macroscopic kinetic equations which describe the photochemical processes of PDT, we can express the reacted 1O2 concentration, [1O2]rx, in a form related to time integration of the product of 1O2 quantum yield and the PDT dose rate. The production of [1O2]rx involves physiological and photophysical parameters which need to be determined explicitly for the photosensitizer of interest. Once these parameters are determined, we expect the computed [1O2]rx to be an explicit dosimetric indicator for clinical PDT. Incorporating the diffusion equation governing the light transport in turbid medium, the spatially and temporally-resolved [1O2]rx described by the macroscopic kinetic equations can be numerically calculated. A sudden drop of the calculated [1O2]rx along with the distance following the decrease of light fluence rate is observed. This suggests that a possible correlation between [1O2]rx and necrosis boundary may occur in the tumor subject to PDT irradiation. In this study, we have theoretically examined the sensitivity of the physiological parameter from two clinical related conditions: (1) collimated light source on semi-infinite turbid medium and (2) linear light source in turbid medium. In order to accurately determine the parameter in a clinical relevant environment, the results of the computed [1O2]rx are expected to be used to fit the experimentally-measured necrosis data obtained from an in vivo animal model.

Singlet oxygen quantum yields (Φd) in water using beetroot extract and an array of LEDs

It is proposed a simple and inexpensive strategy to determine singlet oxygen (¹O2) quantum yields (ΦΔ) of photosensitizers (PS) in water using beetroot extract containing betacyanin (Bc) and a set of light emitting diodes (LEDs) for excitation. Bc, a cationic natural dye, was obtained by flash chromatography purification from the red beet extract (Beta vulgaris) and employed as a convenient probe for ¹O2 detection. Solutions of Bc and PS were illuminated with an array of LEDs adapted in the cuvette compartment of a commercial spectrophotometer, and the decrease in Bc absorbance was followed as a function of time. Bc photobleaching decreased in de-aerated solution and increased in D2O, indicating the involvement of ¹O2. The observed photobleaching rate constant (k obs) was proportional to the LED intensity, concentration and ΦΔ of the PS. By keeping the light source constant we could estimate the overlap integral (R) between the LED emission and PS absorbance for different PS concentrations. The slope of R versus k obs is the value of the photobleaching rate constant (k), which was shown to be proportional to ΦΔ. Values of ΦΔ obtained by this method were compared with those obtained by measuring NIR (near infrared) emission for a series phenothiazine dyes.

SINGLET OXYGEN QUANTUM YIELDS OF PORPHYRIN-BASED PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY

The major cytotoxic agent with most current photosensitizers used in photodynamic therapy (PDT) is widely believed to be singlet oxygen (1O2). Determination of the1O2quantum yields for porphyrin-based photosensitizers, including hematoporphyrin derivative (HiPorfin), hematoporphyrin monomethyl ether (HMME) and photocarcinorin (PsD-007) in air-saturated dimethylformamide (DMF) solutions were performed by the direct measurement of their near-infrared luminescence. In addition,1O2quencher sodium azide was employed to confirm the1O2generation from the investigated photosensitizers. The maximal1O2luminescence occurs at about 1280 nm with full width at half maximum of 30 nm. The1O2quantum yields were found to be 0.61 ± 0.03, 0.60 ± 0.02 and 0.59 ± 0.03 for HiPorfin, HMME and PsD-007, respectively. These results provide that these porphyrin-based photosensitizers produce1O2under irradiation, which is of significance for the study of their photodynamic action in PDT.

Generation of Singlet Oxygen via the Composites of Water-Soluble Thiol-Capped CdTe Quantum DotsSulfonated Aluminum Phthalocyanines

Singlet oxygen (1O2), one of the reactive oxygen species, plays an important role in many biomedical applications. The various compounds including the phthalocyanines, quantum dots (QDs) and QD complex, which may have potential to produce 1O2, thus received more and more attentions in recent years. By means of the direct detection of near-infrared 1270 nm, we found that the water-soluble thiol-capped CdTe QDs can photoproduce 1O2 in deuterated water with a low quantum yield (QY) of 1%. When sulfonated aluminum phthalocyanines (AlSPc's) were connected to these QDs, forming water-soluble QD-Pc composites, the 1O2 QY of the composites increased to 15% under the excitation of 532 nm, while little 1O2 production can be found for AlSPc alone at the same excitation because of the poor absorption of AlSPc in this region. The results of indirect measurements of 1O2, obtained from the photodegradation of the 1O2 chemical trap anthracene-9,10-diyl-bis-methylmalonate (ADMA), confirmed 1O2 yields in both QD and QD-Pc composite solutions. The QD-Pc composites have the advantage of extending the excitation region to 400-600 nm with remarkably enhanced extinction coefficients as compared with that of AlSPc. Therefore QD-Pc composites can fully utilize visible region light excitation to effectively produce 1O2, which may facilitate the applications of QD-Pc composites in broad areas.

Studies on the production of O2(a1Δg,υ= 0) and O2(b1Σg+,υ= 0) from collisional removal of O2(A3Σu+,υ′ = 6-10)

[1] Production of two metastable O2 species, O2(a1Δg, υ = 0) and O2(b1Σg+, υ = 0), from collisional removal of O2(A3Σu+) by O2 and N2 is investigated at a temperature of 240 K. A state-selective two-laser technique is used, in which the output of the first laser excites ground-state O2 molecules to a specific vibrational level (6 ≤ υ′ ≤ 10) in the A3Σu+ state, and the output of the second laser subsequently probes the O2(a1Δg, υ = 0) and O2(b1Σg+, υ = 0) populations by resonance-enhanced multiphoton ionization via the intermediate d1Πg Rydberg state. For the relatively short timescales of our experiment (∼10−4 Torr·s), we find that the O2(a1Δg, υ = 0) quantum yield from collisional removal of O2(A3Σu+, υ′) by O2 is approximately constant for υ′ = 7–9, and decreases by 30–40% for υ′ = 6 and 10. The O2(b1Σg+, υ = 0) yield is nearly constant for υ′ = 8–10, and decreases by about 50% for υ′ = 6 and 7. The eventual O2(a1Δg, υ = 0) and O2(b1Σg+, υ = 0) yields from O2(A3Σu+, υ′ = 8) measured in synthetic air are nearly equal to those measured in pure O2, suggesting that N2 collider does not significantly affect the relaxation pathways leading to the production of the two metastable species. However, at short experimental times the buildup of the O2(a1Δg, υ = 0) population is considerably slower in synthetic air than in O2.

Spectroscopic Studies and Photodynamic Actions of Hypocrellin B in Liposomes¶

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg l-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB·−) to oxygen. The disproportionation of O2·− can generate H2O2 and ultimately the highly reactive ·OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2·− directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2·−) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.

Dihydrocercosporin Singlet Oxygen Production and Subcellular Localization: A Possible Defense Against Cercosporin Phototoxicity in Cercospora

Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents. We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (phi SO = 0.14-0.18) and very poor in water (phi SO = 0.02-0.04). By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (phi SO = 0.84-0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.

α-Methylstilbene and the Duality of Mechanism in the Quenching of Stilbene Triplets by Molecular Oxygen†

Spin-exchange quenching of alpha-methylstilbene triplets by molecular oxygen and by the free radical di-tert-butyl nitroxide is shown to favor the cis isomer more than does natural decay. The effect of the two quenching events is an identical 7% decrease in the fraction of perpendicular triplets that decay to the trans isomer. The conclusion that relaxed stilbene triplets reside in a shallow minimum corresponding to a geometry in which the two benzyl moieties are orthogonal was based on the observation that their quenching by O2 does not alter the trans/cis photostationary ratio. Our results with alpha-methylstilbene confirm the hypothesis that in the case of stilbene spin exchange quenching by O2 at the twisted geometry favors the cis isomer but occurs in competition with excitation transfer from transoid triplets that leads to the trans isomer and to singlet oxygen. The opposite effects of the two oxygen quenching paths on stilbene isomer composition cancel accidentally leading to an overall insensitivity of benzophenone-sensitized photostationary states to the presence of oxygen. Quenching rate constants derived on the basis of this cancellation are close to diffusion-controlled and predict singlet O2 quantum yields of 0.08 and 0.13 in the presence of air and under an O2 atmosphere, in good agreement with experimental measurements.

Self‐sensitized Photodegradation of Membrane‐bound Protoporphyrin Mediated by Chain Lipid Peroxidation: Inhibition by Nitric Oxide with Sustained Singlet Oxygen Damage

ABSTRACTIn the presence of exciting light, iron and reductants, the singlet oxygen (1O2)‐generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain‐breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX‐containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5α‐OOH and 7α/β‐OOH as respective 1O2 and free radical reporters. 5α‐OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7α/β‐OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7α/β‐OOH buildup as well as 5α‐OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5α‐OOH slowdown. Accompanying the 5α‐OOH effect was a steady decrease in 1O2 quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO‐inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5α‐OOH‐bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely 1O2‐mediated damage. Supporting this was our observation that 1O2‐mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5α‐OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX‐sensitized free radical damage, but PpIX itself, thereby extending its 1O2‐generating lifetime. Consistent findings were obtained using porphyrin‐sensitized COH‐BR1 cells. These previously unrecognized effects of NO could have important bearing on 5‐aminolevulinate‐based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.

Self-Sensitized Photodegradation Of Membrane-Bound Protoporphyrin Mediated By Chain Lipid Peroxidation: Inhibition By Nitric Oxide With Sustained Singlet Oxygen Damage

In the presence of exciting light, iron and reductants, the singlet oxygen (1O2)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5alpha-OOH and 7alpha/beta-OOH as respective 1O2 and free radical reporters. 5alpha-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7alpha/beta-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7alpha/beta-OOH buildup as well as 5alpha-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5alpha-OOH slowdown. Accompanying the 5alpha-OOH effect was a steady decrease in 1O2 quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO-inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5alpha-OOH-bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely 1O2-mediated damage. Supporting this was our observation that 1O2-mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5alpha-OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX-sensitized free radical damage, but PpIX itself, thereby extending its 1O2-generating lifetime. Consistent findings were obtained using porphyrin-sensitized COH-BR1 cells. These previously unrecognized effects of NO could have important bearing on 5-aminolevulinate-based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.