WAVELENGTH-DEPENDENT EFFECTS OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A in vivo AND in vitro

Abstract Benzoporphyrin derivative monoacid ring A (BPD-MA) is a chlorin-like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P8 15 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon-ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBN2 mice injected with 2 mg/kg of BPD-MA and exposed to light at 125 J/cm², between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630–640 nm and 680–690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680–690 nm caused slightly more damage to normal skin than light delivered at 630–640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 68–95 nm. Antitumor efficacy in tumor-bearing DBN2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor-free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm. When tumor-bearing animals injected with BPD-MA at 2 mdkg and exposed to light 3 h later were treated with either 630 or 690 nm light at various doses, it was observed that 690 nm light was more effective at tumor ablation than was 630 nm light, demonstrating that while similar damage to normal skin may be effected by equivalent doses of light at either wavelength, tumor ablation was greater at 690 nm. Further, our data suggest that alternative light sources with bandwidths greater than those of the argon-ion pumped dye laser (±0.3 nm) may have equivalent efficacy with this photosensitizer.     

LIPOSOMAL DELIVERY OF A PHOTOSENSITIZER, BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD), TO TUMOR TISSUE IN A MOUSE TUMOR MODEL

Abstract— Biodistribution studies were carried out on ¹⁴C-labeled benzoporphyrin derivative monoacid ring A (BPD), which had been formulated as a unilamellar liposome or taken from a stock solution in dimethyl sulfoxide diluted into phosphate-buffered saline immediately before intravenous injection into DBA/2 mice. By and large the general distribution of BPD to various organs and tissues was comparable for both formulations. It was noted, however, that liposomal material appeared to enter tissues more rapidly and to be cleared more rapidly, as demonstrated by shorter half-lives for a number of tissues including skin, lung and fat, and generally lower levels in most tissues 24 h following administration. Accumulation in tumor tissue was slightly higher with liposomal BPD, and clearance rates for this tissue were equivalent (half-lives 16.1 h for liposomal BPD and 16.9 h for aqueous BPD). When the two preparations were tested in a bioassay in tumor-bearing mice, photodynamic therapy (PDT) with liposomal BPD proved to be superior to the aqueous preparation when PDT was administered 3 h following intravenous administration of BPD. Plasma distribution studies in vitro demonstrated that 91.1 ± 0.3% of the liposomal BPD distributed to the lipoprotein fraction within the first hour of mixing, whereas only 49.1 ± 2.6% of nonliposomal BPD was associated with lipoprotein under the same conditions. Furthermore, while lipoprotein-associated liposomal BPD distributed evenly between all three types of lipoprotein (high, low and very low density), a majority of nonliposomal BPD associated with the high-density lipoprotein fraction.     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF 2-AMINO-4 PTERIDINONE: A NATURAL PIGMENT

Abstract— The characteristics of the fluorescence and phosphorescence emission of 2-amino-4 (3H) pteridinone (or pterin) in aqueous solutions are pH dependent. The room temperature fluorescence quantum yield is low and is maximum at pH = 10 (φ_F∼ 0.057). The 77K phosphorescence emission consists of two overlapping emissions originating from τ* triplet states. In agreement with low temperature results, the 353nm laser flash photolysis makes it possible to detect at pH 9.2, two transient triplet absorptions (τ₁∼ 0.3 μs and τ₂∼ 2.3 μs). The longer lived triplet is characterized by φ_TM∼ 0.20 and ∼ (550nm) = 2000 M ⁻¹ cm⁻¹. It reacts with the solvent forming the semireduced pterin with a quantum yield φ_R∼ 0.06. The photosensitizing properties of pterin have been studied by laser flash spectroscopy and steady state irradiations. Photoreactions implying singlet oxygen formation are shown to occur. Laser flash spectroscopy indicates that the pterin triplet is reduced by amino acids and nucleic acid bases. Corresponding bimolecular reaction rate constants have been measured.     

聚对苯撑乙烯类共聚物/TiO2复合材料光物理性能的研究

两种经过化学修饰的PPV(聚对苯撑乙烯)类共轭高分子共聚物分别与纳米TiO₂复合,作为有机/无机复合材料进行研究.这两种共聚物在乙醇、二氯甲烷溶液中分别与以Sol-Gel法、反胶束法制得的TiO₂共混得到均匀分散的体系而不出现相分离.用共聚物与TiO₂的复合液可以在石英基底上制成均匀的复合膜.结果表明,高聚物/TiO₂复合物的光物理特性与单纯的高聚物相比呈现明显的差异;不同粒径的纳米TiO₂对复合物性能的影响不同;中间苯环的取代基对共聚物的性质影响明显.共轭高分子与纳米TiO₂复合涂膜后其发光性能明显改善,有作为发光器件的应用前景.     

PHOTOPHYSICAL AND PHOTOBIOLOGICAL PROPERTIES OF DIPORPHYRIN ETHERS

Spectral properties of several diporphyrin ethers were assessed in different solvents and after accumulation by leukemia L1210 cells in vitro. To facilitate studies in a variety of solvents, both tetramethylesters of the diporphyrin ethers and free acids were employed. For comparison, studies on the corresponding porphyrin monomers were also carried out. The joining of two porphyrins by an ether linkage had several consequences. We observed a blue shift in the Soret band of the ethers, but not of the corresponding simple porphyrins, in protic solvents. This phenomenon is likely related to ether aggregation under conditions which promote H-bonding. The presence of an ether linkage was associated with enhanced fluorescence at 630-640 nm and decreased fluorescence lifetimes and yields, especially in protic solvents. The ether linkage was unaffected by intracellular enzymes, but porphyrin esters were readily hydrolyzed upon accumulation by L1210 cells. The joining of two hematoporphyrin molecules by an ether linkage promoted dye accumulation by L1210 cells. In contrast, accumulation of mesoporphyrin and protoporphyrin was thereby retarded.     

In vitro PHOTOSENSITIZATION WITH A BENZOPORPHYRIN DERIVATIVE

Biophysical and photobiological properties of two benzoporphyrin derivatives were examined. These dyes exhibit substantial absorbance in the red, and are potent photosensitizers in vitro. After brief (0.5 h) incubations, phototoxicity was more closely correlated with membrane than with mitochondrial photodamage. Affinity of these dyes toward plasma lipoproteins are consistent with a mode of localization via the LDL-mediated mechanism utilized by the hematoporphyrin-derived product, HPD.     

ON THE PHOTOSENSITIZING PROPERTIES OF N-FORMYLKYNURENINE AND RELATED COMPOUNDS

Abstract— The photochemical and photosensitizing properties of N -formylkynurenine (FK) and related compounds have been investigated using the laser flash photolysis technique by exciting water solutions with 265 nm or 353 nm radiation. The FK molecules in their first excited singlet state readily react with water leading probably to OH formation. FK triplet state reacts with many biological compounds including vitamins, amino acids and nucleic acid bases. The semi-reduced FK thus formed can, in turn, reduce substrates such as cytochrome c or O₂.     

PHOTOSENSITIZING EFFECTS OF HEMATOPORPHYRIN DERIVATIVE IMMOBILIZED ON SEPHAROSE

Abstract— The cytotoxicity that ensues following photosensitization by hematoporphyrin derivative (Hpd) is attributed to production of singlet oxygen. Many of the cellular end points reported to be affected are localized to membranes, hydrophobic environments conducive to partitioning of hydrophobic porphyrins in Hpd. In order to test the hypothesis that efficacy of Hpd-induced photosensitization is enhanced by its ability to freely enter cells or subcellular organelles, we immobilized Hpd on a sepharose support. This immobilized reagent was found to produce ¹O₂ when photoradiated, in yields similar to those observed for Hpd in solution, as evidenced by the bleaching of p -nitrosodimethylaniline in the presence of imidazole. The immobilized Hpd was capable of photosensitizing, i.e. inhibit, cytochrome c oxidase activity in intact mitochondrial membranes and in aqueous solution. However, enzymes located on the interior of mitochondrial membranes (F₀F₁ ATP synthase and succinate dehydrogenase), in the mitochondrial matrix (malate dehydrogenase), or on the inside of the plasma membrane, (Na++ K+)- ATPase, were unaffected by immobilized Hpd plus photoradiation compared to free Hpd. The results suggest that photosensitization by Hpd most likely arises from entry of the photosensitizer into the biological membrane, although proteins on the exterior membrane surface may be susceptible to damage by ¹O₂ produced in proximity to their location.     

In vitro AND in vivo UPTAKE OF BENZOPORPHYRIN DERIVATIVE INTO HUMAN AND MINISWINE ATHEROSCLEROTIC PLAQUE

Abstract— Befnzoporphyrin derivative(BPD) has been demonstrated to be fnew potent photosentsitze for photodynamic therapy(PDT). Althought most of wrok on BPD has been focused on its potential applications for cancer tratment, BPD amy have potential clilnical uses in the treatment of artheros clerosis. The purposes of this study was to determine in vitro and vivo uptake of BPD into atherosclerotic plaque. Samples of atherosclerotic human femoral and popliteal arteries were incubated with BPD-monoacid, ring A(BPD-MA) for 1 h in the following concentrations: 1, 5, 10, 20, 30 and 40 μg/mL. fluorescence from all samplesd was determined by chemical etraction with a spectrofluorometer. the tissue concentration for human arteries was 0.37 ± 0.03, 2.78 ± 1.5, 3.6 ± 1.91, 7.15 ± 2.36, 8.06 ± 3.09 and 14.6 ± 4.81 μg/g, respectively. In aeddition, three miniswine were rendered atherosclerotic and given BPD 2.0 mg/ Kg intravenously. The concentration of BPD-MA in miniswine aorta was93–190 ng/g and the plaque/normal ration was 1.7–3.5, for miniswine cartoid artery contained 54 ng/g. this study showed that BPD-MA was taken up in atherosclerotic vesselsd both in vitro and in vivo and mey have potential for PDT of atherosclerosis.     

PHOTOPHYSICAL PROPERTIES AND CONFORMATIONAL EQUILIBRIUM OF trans-6-STYRYLQUINOXALINE

The wavelength dependent fluorescence spectra and bi-exponential fluorescence decays of trans-6-styrylquinoxaline (6-StQx) indicate that two conformers exist in equilibrium in solution. When comparing the conformationally restricted analogues (5Me-6-StQx and 7Me-6-StQx) with 6-StQx, the short-wavelength absorbing conformer of 6-StQx can be assigned to conformer(A) and the long-wavelength absorbing component to conformer(B). The temperature effects on the fluorescence lifetime indicate that the short-lived conformer(A) is the more stable conformer in non-polar solvents, while the long-lived conformer(B) is the stable component in polar solvents. The results of semiempir-ical AMI and CNDO/S CI calculations on both the ground and excited states are consistent with the conclusions drawn from the spectral data.     

SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF POLYMER-BOUND HEXANUCLEAR MOLYBDENUM CLUSTERS

Abstract

The coordination of [Mo₆Cl₈ ⁱ(SO₃CF₃)₆ ^a]^2? (1) and [Mo₆Cl₈ ⁱCl₄ ^a(EtOH)₂ ^a] (2) to poly(evinylpyridine) (PVP) produces crosslinked materials. Formation of these materials occurs by displacement of axial ligands ((a) in Fig. 1) SO₃CF₃ ^? or EtOH on the clusters by the vinylpyridine moieties. The availability of six coordination sites in 1 and two coordination sites in 2 offers control over the degree of crosslinking and solubility of the materials. Similarly, varying the ratio of cluster coordination sites (C) to polymer-pendant pyridine ligands (P) yields materials with a wide range of solubilities and glass transition temperatures (T_g). Materials with C:P ratios of 1:100 are highly soluble in CH₂Cl₂ and MeOH, with T_g values slightly higher than that of pure PVP. Unswellable materials with no discernible T_g are obtained with C:P ratios of 1:5 and lower, indicating a high degree of crosslinks. Transient emission spectroscopy reveals that polymer-bound cluster 1 lacks the luminescent properties characteristic of many [Mo₆Cl₈]⁴⁺ based clusters. Conversely, polymer-bound cluster 2 displays intense luminescence and retains the unusually long-lived excited-state observed for the free clusters in solution. The emission lifetime of PVP-bound 2 fits a biexponential decay, with τl₁ = 90 μs (60 %) and τ₂ = 8 μs (40 %), while [Mo₆Cl₈ ⁱCl₆ ^a]^2? has a uniexponential decay of τ = 156 μs in CH₂Cl₂ solution.     

含烷基侧链聚芴基苯并二噁唑聚合物的合成及性能研究

通过溶液缩聚的方法合成了一种含烷基侧链聚芴基苯并二噁唑聚合物(PBOPFx),讨论了聚合物的光物理性能、热性能和溶解性能.结果表明,在甲基磺酸(MSA)溶液中,PBOPFx紫外吸收显示出最大吸收峰为466 nm左右的蓝光发射,与PBO相比,最大紫外吸收峰和最大荧光发射峰均发生了明显红移,这是由于芴单元的引入,提高了聚合物的共轭程度,加强了对电子的束缚能力.此外,比较了PBOPFx在320 nm处和430 nm处激发得到的荧光发射光谱,预测聚合物中存在两种共轭单元,通过测定氧化还原曲线以及讨论stokes位移,验证了这个假设,但是由于两种共轭单元并没有出现明显的分离现象,同时还伴有电子传输杂化,致使PBOPFx主要显示出共轭长度大的单元的吸收和发射.另外由于芴单元中烷基侧链的存在,聚合物(PBOPFx)的热稳定性远低于聚亚苯基苯并二噁唑聚合物(PBO),起始分解温度在350℃左右,溶解性方面,PBOPFx有了很大的改善,在大多数有机溶剂中能较好溶解.     

共轭刚性苯并二噁唑类聚合物的合成及其光物理性能研究

设计并合成了一系列共轭高分子———聚亚苯基苯并二唑 (PBO) ,聚亚乙烯基苯并二唑 (PBOV)和聚亚丁二唑 (PBODV) ,并对它们进行了表征 .用紫外吸收光谱和荧光光谱对聚合物的溶液和薄膜进行了详细的研究 ,研究结果表明分子结构中双键的引入可以降低聚合物的光带能隙 ,λmax从PBO溶液的 4 2 8 5nm (Eg=2 76eV) ,到PBOV溶液的 4 80 5nm (Eg=2 4 6eV) ,再到PBODV溶液的 4 96 0nm (Eg=2 38eV) .共轭程度的增加使得聚合物溶液更容易发生集聚从而导致荧光自熄灭 .     

吡唑啉类化合物在溶剂中的溶致变色和光物理

在合成了一系列取代的二苯基吡唑啉衍生物基础上对其在不同溶剂中的溶致变色行为进行了研究。利用Bilot-Kawaski公式对不同化合物在基态和激发态时分子偶极矩的差值进行了计算, 表明当拉电子基与分子中C-3原子相联时, 化合物的偶极矩可发生很大变化, 说明拉电子基团的引入有力地促进了分子内的电荷转移。用E_T(30)值作为不同溶剂极性大小的指标, 发现它能和该类化合物一系列溶液的光物理性质建立起良好的线性关系。     

ACTIVATION OF BENZOPORPHYRIN DERIVATIVE IN THE CIRCULATION OF MICE WITHOUT SKIN PHOTOSENSITIVITY

In vitro experiments with benzoporphyrin derivative monoacid ring A (BPD) confirmed earlier studies that it was taken up rapidly (within 30 min) to maximum concentrations by all cells tested. It was also confirmed that rapidly dividing tumor cell lines and mitogen-activated murine T lymphocytes took up significantly more (5-10-fold) BPD than did normal splenic lymphocytes. Further experiments were undertaken to determine whether BPD could be activated by whole-body irradiation with red light in the blood of animals, shortly after intravenous (i.v.) administration, in the absence of skin photosensitivity. It was found that shaved and depilated mice injected i.v. 60 min earlier with BPD at between 0.5 and 1.0 mg/kg could tolerate 160 J/cm² of broad-band red light (560-900 nm) delivered, at a relatively low rate, over a 90 min time interval without developing skin photosensitivity or general phototoxicity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 μg/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When LI 210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total-body light treatment (160 J/cm² of 590-900 nm light delivered over 90 min), significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This indicated that sufficient light was being delivered to BPD in the blood flowing in the peripheral vasculature to effect cytotoxicity to cells containing the photosensitizer without causing either vascular or skin photosensitivity. Thus, activation of this photosensitizer in the circulation can be achieved by transdermal light exposure without causing skin photosensitivity provided that light exposure is performed at a time when the first phase of plasma clearance is complete and when the drug has not yet accumulated in skin.     

MIMICRY OF CAROTENOID FUNCTION IN PHOTOSYNTHESIS: SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF A CAROTENOPYROPHEOPHORBIDE

Abstract— A new molecule consisting of a carotenoid polyene covalently linked to a pyropheophorbide has been prepared. This molecule exists in solution in an extended conformation with the carotenoid moiety relatively far from the tetrapyrrole π-electron system. The compound shows negligible antenna function (singlet energy transfer from carotenoid to pyropheophorbide), but substantial photoprotection from singlet oxygen production (due to rapid triplet energy transfer from pyropheophorbide to carotenoid). The photochemical properties of this molecule parallel those of conformationally similar carotenoporphyrins. Thus, carotenoporphyrins appear to be valid synthetic systems for modeling in vivo carotenoid-chlorophyll interactions.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

PHOTOPHYSICAL AND PHOTOCHEMICAL PROPERTIES OF TRITON X-165 IN AQUEOUS AND METHANOLIC SOLUTIONS

Abstract Laser flash photolysis of the nonionic surfactant Triton X-165 was carried out at 248 nm in methanolic and aqueous solutions at different pHs. Cation radical, triplet, phenoxy radical and solvated electron were observed as the transient absorbing species. Various characteristics of these transient species and their decay constants are reported. Photoionization was found to be a major process and it was monophotonic. The results obtained are compared with the photolysis of a small molecule p -methoxytoluene (PMT). Fluorescence and phosphorescence properties of the surfactants Triton X-100, Triton X-165 and PMT are also studied. A suitable reaction scheme is proposed to account for the observed results.     

SENSITIVITY OF ACTIVATED MURINE PERITONEAL MACROPHAGES TO PHOTODYNAMIC KILLING WITH BENZOPORPHYRIN DERIVATIVE

Abstract— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-γ (rIFN-γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-γ-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD₅₀= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm²) than cells (LD₅₀= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-γ-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD₅₀= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-γ-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN-γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.