TUMOR CELL SPECIFIC DARK CYTOTOXICITY OF LIGHT-EXPOSED MEROCYANINE 540: IMPLICATIONS FOR SYSTEMIC THERAPY WITHOUT LIGHT

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment. The observed cytotoxicity of light-exposed MC540 to the tumor cells was significantly greater (P less than 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light-exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia-bearing mice treated with light-exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light-exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light-exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short-lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.     

PHOTOCHEMISTRY OF MEROCYANINE 540

The photophysical properties of merocyanine 540 have been determined in methanol solution over a modest temperature range. Triplet state population is inefficient (the limiting triplet quantum yield being 0.25) due to rapid isomerization of the central double bond from the first excited singlet state. Activation energies have been measured for isomerization from the excited singlet state (20 kJ mol-1) and for conversion of the resultant cis-isomer back to the original trans-form (63 kJ mol-1), both processes involving formation of a twisted species. The dye is easily oxidized to give an unstable adduct which decomposes on the sub-ms timescale. Reversible redox chemistry occurs upon excitation in the presence of electron acceptors. These various observations are discussed in terms of the known chemotherapeutic activity of MC540 and it is concluded that the most probable mechanisms for cytotoxicity involve either local thermal disruption of cell membranes or in situ photogeneration of toxins derived from breakdown of the dye.     

POTENTIATION OF MEROCYANINE 540-MEDIATED PHOTODYNAMIC THERAPY BY SALICYLATE and RELATED DRUGS

Abstract— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.     

ANTIVIRAL ACTIVITY OF MEROCYANINE 540

Abstract Simultaneous exposure to the lipophilic dye merocyanine 540 (MC 540) and white light inactivates several enveloped viruses. The same treatment appears to have little or no effect on pluripotent hematopoietic stem cells, mature red cells, and mature leukocytes. At least some components of the clotting system are spared, too. The molecular basis of the virucidal effect of MC 540 and light is not yet completely understood. Based on what is known about the interactions of MC 540 with cells and artificial membranes, it seems likely that MC 540 binds to and damages the viral envelope. MC 540-mediated photosensitization may have implications for the sterilization of bone marrow and blood products, the preparation of vaccines, and selected areas of antiviral therapy.     

DIFFERENTIAL INACTIVATION OF SURROGATE VIRUSES WITH MEROCYANINE 540

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, phi X174, T7, PRD1, and phi 6, by the dye merocyanine 540 (MC540) (15 micrograms/mL). Merocyanine 540 (MC540) should be most effective with lipid-containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and phi 6 contain lipid, with only phi 6 having an external lipoprotein envelope. Filtered radiation (450-600 nm) from a 750 W projector was used at 16-100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with phi 6 (Do = 1.5 kJ/m2) being the most sensitive, followed by T7 (21-fold less sensitive). While both PRD1 and phi 6 have lipid components, only phi 6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do = 0.053 kJ/m2) and found it to be 28-fold more sensitive than phi 6 to photoinactivation by the same concentration of MC540. Thus phi 6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.     

PHOTOBLEACHING OF MEROCYANINE 540: INVOLVEMENT OF SINGLET MOLECULAR OXYGEN

The purpose of this study was to assess the mechanism of merocyanine 540 (MC540) photobleaching in a liposomal system. Broad based visible irradiation of MC540 in unilamellar dilauroylphosphatidylcholine (DLPC) vesicles resulted in dye bleaching that was strictly O2 dependent. The rate of self-sensitized photobleaching was enhanced in D2O and inhibited by both azide and histidine, consistent with 1O2 intermediacy (Type II chemistry). Supportive evidence for this mechanism was obtained by using a Type II sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS lambda max = 678 nm). Irradiation of AlPcS and MC540 in DLPC with lambda greater than 630 nm (absorbed only by AlPcS) light resulted in rapid bleaching of MC540, which was stimulated by D2O and inhibited by azide. A rate constant of 10(7) M-1 s-1 was determined for the chemical quenching of 1O2 by MC540. The rate constant for physical quenching of 1O2 by MC540 was estimated to be ca 10(9) M-1 s-1.     

TRIPLET YIELD OF MEROCYANINE 540 IN WATER IS WAVELENGTH DEPENDENT

Monomers and aggregates of Merocyanine 540 (MC540) in water are able to photoisomerize. The shape of the photoisomer absorption spectrum is very similar to that of the ground state. Triplet state of MC540 in water has been produced by energy transfer from triplet anthracene and displays a broad absorption spectrum between 600 and 700 nm. The triplet state may also be produced by direct excitation of MC540 with UV light. However, when the dye is excited by visible light, no triplet state absorbance in the red could be detected so that the triplet yield of MC540 in water seems to be excitation wavelength dependent.     

ACTION SPECTRA OF THE ANTILEUKEMIC and ANTIVIRAL ACTIVITIES OF MEROCYANINE 540

Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.     

MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF LEUKEMIA CELLS: EFFECTS OF DOSE FRACTIONATION

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.     

生理条件下对苯二酚的电化学行为及电极产物的色谱鉴定

用循环伏安法研究了对苯二酚在玻碳电极上的电化学行为,从循环伏安图上看出当在0至1．6V电位间连续扫描20次,在0．63V左右可见到一氧化峰电位,其峰电流随扫描次数的增加而减小。说明电极反应为不可逆过程。然而,如改变在-1．4～1.6V电位间扫描,仅数次扫描后即可在Epa=0．25V和Epc=-0．08V处见到一对氧化还原峰电位,说明了此过程为一可逆过程。同时,对电极反应的产物用RHPLC进行研究和鉴定,从色谱图所示结果与电化学法所得结果一致。根据此工作的研究结果对苯二酚在呼吸链中所产生的作用进行了探讨。     

LIMITED CELL-CYCLE DEPENDENCE OF THE MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF L1210 LEUKEMIA CELLS

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.     

In vivo TUMOR OXYGEN TENSION MEASUREMENTS FOR THE EVALUATION OF THE EFFICIENCY OF PHOTODYNAMIC THERAPY

Among the sequence of events which occur during photodynamic therapy (PDT) are depletion of oxygen and disruption of tumor blood flow. In order to more clearly understand these phenomena we have utilized transcutaneous oxygen electrodes to monitor tissue oxygen disappearance. These results provide, for the first time, non-invasive real-time information regarding the influence of light dose on tissue oxygenation during irradiation. Measurements were conducted on transplanted VX-2 skin carcinomas grown in the ears of New Zealand white rabbits. Rabbits were treated with Photofrin II and tumors were irradiated with up to 200 kJ/m2 (500 W/m2) of 630-nm light. Substantial reductions in tumor oxygen tension were observed upon administration of as little as 20 kJ/m2. For a series of brief irradiations, oxygen tension was modulated by the appearance of laser light. Tissue oxygen reversibility appeared to be dependent upon PDT dose. Long-term, irreversible tissue hypoxia was recorded in tumors for large (200 kJ/m2) fluences. These results suggest that transcutaneous oxygen tension may be useful as a general indicator of the effectiveness of PDT and as an in situ predictor of the energy required to elicit tumor damage.     

THE MICROVASCULAR EFFECTS OF PHOTODYNAMIC THERAPY: EVIDENCE FOR A POSSIBLE ROLE OF CYCLOOXYGENASE PRODUCTS

Photodynamic therapy (PDT) of malignant tumours may involve the interruption of tumor and peritumor microcirculation. We have studied the effect of light activation of the photosensitizing drug dihematoporphyrin ether (DHE) on rat subcutaneous arterioles and the modulation of these effects by cyclooxygenase inhibitors indomethacin and acetyl salicylic acid (ASA). Animals received DHE 48 h prior to light activation and additionally either indomethacin, ASA or saline 3 h prior to treatment. Light activation (630 nm, 60 J/cm2) resulted in a significant reduction to 62 +/- 2% SEM of initial blood flow. This effect was inhibited by ASA (98 +/- 8% SEM) and indomethacin (87 +/- 8% SEM). Results from the administration of various doses of both compounds indicate that this inhibition is dose related. The data presented here show that PDT causes a significant reduction in blood flow in normal arterioles and that this effect was inhibited by ASA and indomethacin indicating that prostaglandins or thromboxane A2 may play an important role in the microvascular response to PDT.     

温和条件下羰基合成的新方法

介绍了以烯烃、卤代烃、烷烃及芳烃为底物 ,在光促进和温和条件下 ,用过渡金属催化进行羰基合成的新方法。试验结果表明 ,上述底物用这种新方法进行羰基合成 ,反应的选择性好 ,其中烯烃和卤代烃的收率也较高。可以预计这种新的羰基合成方法的研究具有一定的理论和应用价值。     

可溶性多氟烷氧基取代金属酞菁 :新型肿瘤光动力治疗光敏剂(英文)

合成了 9个新的多氟烷氧基取代的金属酞菁衍生物 ,它们均可溶于多数有机溶剂 .光氧化能力和对DNA的光切断活性的研究表明 ,中心金属离子为锌和铝的酞菁衍生物 ,具有较好的产生单重态氧的能力 .在乳化剂F68的存在下 ,锌酞菁衍生物 2a对离体杂交瘤细胞表现出良好的光灭活作用     

THE PHOTOPHYSICS OF MEROCYANINE 540. A COMPARATIVE STUDY IN ETHANOL AND IN LIPOSOMES

Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> ø_T > 0.02 in ethanol and 0.01 > ø_T- > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay.     

AN ELECTRON SPIN RESONANCE STUDY OF MEROCYANINE 540-MEDIATED TYPE I REACTIONS IN LIPOSOMES

The merocyanine 540 (MC540)-mediated reduction of nitroxide spin labels in a liposomal system was examined using electron spin resonance (ESR) spectroscopy. Spin label reduction was light driven, and occurred in liposomes composed of both fully-saturated (dimyristoyl) and mono-unsaturated (1-palmitoyl-2-oleoyl) phosphatidylcholine. Loss of the nitroxide ESR signal was enhanced by the physiological electron donors glutathione, cysteine, and NADPH; and was strongly inhibited by the presence of molecular oxygen. Nitroxides reduced in the presence of MC540 alone could be regenerated either by purging the sample with air or by the addition of ferricyanide, indicating that the ESR signal loss was due to reduction to the corresponding hydroxylamines. Only partial regeneration was attained for nitroxides reduced in the presence of glutathione, cysteine, or NADPH. Reduction rates for the lipophilic spin labels, 5-, 12-, and 16-doxyl stearic acid, were not influenced by the position of the nitroxide moiety along the alkyl chain, however reduction of spin labels occupying primarily the aqueous phase was much slower. These studies demonstrate that MC540 can initiate oxidation/reduction (Type I) reactions. Such Type I processes may augment the effects of singlet oxygen in MC540-mediated photodynamic therapy.     

A STUDY ON THE DEGRADATION MECHANISM OF PHOTOCROSSLINKING PRODUCTS FORMED BY CYCLIZED POLYISOPRENE-DIAZIDE SYSTEM UNDER THE INFLUENCE OF ALKYL BENZENE SULFONIC ACIDS

The degradation mechanism of photocrosslinking products formed by cyclized polyisoprene-diazide system under the influence of the different alkyl benzene sulfonic acids was studied. The effects of alkyl chain length and the concentration of alkyl benzene sulfonic acids on the rate of degradation reaction were discussed. It was found that in the initial stage of degradation, the cyclicity ratio and the average fused ring number did not change considerably, but the percentage of uncyclized parts content varied significantly. The suitable mechanism was supposed.     

试样两端固定或松弛态结晶对取向非晶态聚对苯二甲酸乙二酯膜结晶的影响

The Crystallization of constrained and fre oriented amorphous PET films were studied by using density, birefringence and stress measurements. It has been shown that depending on the birefringence of original sample, the effect of fixing two ends of samples on the Crystallization rate shows different behavior. For amor hous PET film with high birefringence the Crystallization rate of constrained sample is faster. For oriented amorphous PET with very low birefringence the Crystallization rate of free sample is faster.     

试样两端固定或松弛态结晶对取向非晶态聚对苯二甲酸乙二酯膜结晶的影响

<正> 在研究聚合物的结晶过程中,有时(如在同步辐射法、X-衍射法和红外法的测定中)须将试样两端固定,有时(如研究热处理过程中取向聚合物的尺寸和结构变化)须将试样处于松弛状态。因而研究两端固定或松弛状态对取向聚合物结晶速率和结构的影响引起了人们的兴趣。一些研究结果得出,与松弛状态结晶相比,试样两端固定态结晶使取向聚合物的结晶速率变慢,但有时也出现相反的结果。此外,在上述文献中所用的原始试样都已具有较高的结晶度。为了弄清这一问题,我们用密度、双折射和应力测量等方法对取向程度不同的非晶态PET膜进行了深入和系统的研究。结果表明,试样两端固定与否对结晶速率的影响与试样原始的取向程度有关。