POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY—II. PHOTOPHYSICAL PROPERTIES OF [26] PORPHYRIN

Abstract— –Photophysical properties of [26] porphyrin (26 P) were investigated in chloroform. The quantum yields of fluorescence, of S₁→ T₁ intersystem crossing and singlet oxygen formation were measured. The purity, stability, the strong absorption in the red (δ_max= 783 nm; ε_max= 28 000 M ¹ cm^-1) and the ability of singlet oxygen formation recommend 26 P as potential photosensitizer for tumor therapy.     

CHLORIN AND PORPHYRIN DERIVATIVES AS POTENTIAL PHOTOSENSITIZERS IN PHOTODYNAMIC THERAPY

In order to find a photosensitizer with better optical properties and pharmacokinetics than Photofrin II, a series of new photosensitizers related to methyl pheophorbide-a and chlorin-e6 were synthesized. These compounds absorb at substantially longer wavelengths (lambda max 660 nm) than does Photofrin II (630 nm) and show promise for use in photodynamic therapy. Among the porphyrins, we observed that long carbon chain ether derivatives are better photosensitizers than their ester analogs. These sensitizers were tested for in vivo photosensitizing activity vis-a-vis Photofrin II, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. Most of these photosensitizers were found to have better tumoricidal photosensitizing activity than Photofrin II and demonstrated more rapid attenuation of normal tissue photosensitivity with time after administration vis-a-vis Photofrin II.     

THE PHOTOPHYSICAL PROPERTIES OF PORPHYCENES: POTENTIAL PHOTODYNAMIC THERAPY AGENTS*

Porphycene and a tetra-n-propyl derivative remained unaltered on irradiation in toluene at room temperature. Quantum yields of fluorescence, S T intersystem crossing, and singlet molecular oxygen sensitization, as well as lifetimes of the singlet and triplet excited states were measured. In view of their structural relationship to porphyrin, their high absorption above 620 nm, their stability towards photooxidation, and their high quantum yields of fluorescence and singlet oxygen sensitization, these compounds qualify as potential agents for tumor marking and photodynamic therapy.     

PHOTOPHYSICAL PROPERTIES OF A CHLORIN, A POTENT SENSITIZER FOR PHOTOCHEMOTHERAPY

Abstract— Photophysical and photodynamic properties of a ehlorin type molecule derived from hydroxyethylvinyldeuteroporphyrin are presented. It photosensitizes singlet oxygen production as efficiently as mesotetraphenylporphin. The high absorptions of both its ground and triplet states in the red (660 nm) make it a potent photosensitizer which might act not only by photo-oxidation via singlet oxygen but also by radicals produced via sequential biphotonic absorption.     

A TEST OF DIFFERENT PHOTOSENSITIZERS FOR PHOTODYNAMIC TREATMENT OF CANCER IN A MURINE TUMOR MODEL

Abstract The efficiency of different sensitizers for photodynamic therapy (PDT) was tested using a model system with a C3H mammary carcinoma growing subcutaneously on the dorsal side of mouse feet. Growth curves were constructed from which growth delay and doubling time in the regrowth phase were calculated. As PDT induced oedema in the mouse foot, this model system also allowed assessment of normal tissue response.
The following sensitizers were tested: hematoporphyrin derivative (HpD), Photofrin II (PII), tetraphenylporphinetetrasulfonate (TPPS₄), acridine orange (AO), phthalocyanine tetrasulfonate (PCTS), Al- and Zn-phthalocyanine tetrasulfonate (A1PCTS and ZnPCTS). For tumor control, the following sensitizer efficiencies were found: PII > HpD > AIPCTS > TPPS₄ >>> ZnPCTS, PCTS, AO. With regard to sensitizing normal-tissue damage: PII > AIPCTS, TPPS₄ > HpD, ZnPCTS, PCTS. The results suggest that AIPCTS should be further evaluated for use in PDT.     

OCTA-ALKYL ZINC PHTHALOCYANINES: POTENTIAL PHOTOSENSITIZERS FOR USE IN THE PHOTODYNAMIC THERAPY OF CANCER

The synthesis, characterization and electronic spectra of a series of nine 1,4,8,11,15,18,22,25-octa-alkyl zinc phthalocyanines (ZnPc), potential photosensitizers for the photodynamic therapy of cancer, are described. The substituents on the phthalocyanine (Pc) macrocycle “red-shift” the absorbance maximum, in cyclohexane, of all nine members of this series to a value of 703 ± 2 nm, with a corrected fluorescence emission maximum for the octadecyl derivative of 715 nm. The solubilities and degree of aggregation of six examples in cyclohexane have been measured. The highest homologue, the octadecyl derivative, remains essentially unaggregated to a concentration of 1.5 ± 10^?4 mol dm^?3. The photostability of this Pc has been examined and the compound shown to be sensitive to photooxidation processes, which lead to its decomposition to 3,6-fcw-decylphthalimide. Known singlet oxygen quenchers inhibit the photodecomposition. In a comparative study, the octadecyl ZnPc underwent a more rapid photodecomposition than the corresponding metal-free derivative.     

THE PHOTOSENSITIZERS BENZOPHENOXAZINE and THIAZINES: COMPREHENSIVE INVESTIGATION OF PHOTOPHYSICAL and PHOTOCHEMICAL PROPERTIES

Eight differently substituted title dye compounds have been investigated regarding intersystem crossing, triplet state, fluorescence and singlet excited state pKa properties. In general, non-halogenated oxazines and thiazines as well as a mono bromooxazine show very low triplet quantum yields, phi tau (less than 0.03) and relatively long triplet lifetimes (approximately 40 microseconds) in acidic methanol. The phi tau data correlate well with known singlet oxygen yields. In basic methanol no triplet transient is observed but a significant yield of a ground state transient protonated (base dye) form is produced with a short lifetime, approximately 400 ns. Fluorescence can be seen simultaneously from both the excited base and the protonated base dye forms in basic methanol. For iodinated oxazine or thiazines, the triplet yield increases and can be as high as 0.5 (diiodo case) in acidic methanol. The triplet lifetimes are further shortened to approximately 10 microseconds compared to the non-iodinated derivatives above. The triplet yields of the iodo compounds are higher or equal to known singlet oxygen yields. In basic methanol triplet yields up to 0.2 can be seen, the triplet lifetime are shortened still further to 1 microsecond but no observable protonated form is produced (in distinction to the non-iodinated cases). Consideration is given to the correlation of triplet and singlet oxygen yields, ground and excited pKa properties, spin-orbit coupling and internal conversion properties, solvent effects, and phototherapeutic activity of these dyes.     

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。     

SPECTROSCOPIC PROPERTIES OF POTENTIAL SENSITIZERS FOR NEW PHOTODYNAMIC THERAPY START MECHANISMS via TWO-STEP EXCITED ELECTRONIC STATES

Three substituted tetraazaporphyrins, octa-(4-tert-butylphenyl)-tetrapyrazinoporphyrazine, tetra-(4-tert-butyl)phthalocyanine and tetra-(4-tert-butyi)phthalocyanatomagnesium (t₄-PcMg), were spectroscopically checked in solutions and liposomes with respect to suitability as potential sensitizers of a possible new start mechanism for photodynamic therapy (PDT) from a stepwise excited higher singlet state. This PDT start mechanism was recently proposed to overcome the problem of O₂ (¹δ₂)-caused cutaneous phototoxicity in PDT. By means of absorption and fluorescence measurements as well as nonlinear absorption investigation, transient spectroscopy and lasing experiments, compound t₄-PcMg was found to have the most appropriate properties: Based on a high fluorescence quantum yield (φ_fi= 0.84) and a very low crossing to the triplet (φ_isc= 0.05), two higher excited states can be effectively populated both by two stepwise absorption transitions at 674 nm and consecutive absorption transitions at 674 and 710 nm. Moreover, t₄-PcMg incorporates into liposomes very well with spectroscopic properties similar to those in solution.     

POLYMER BOUND PYRROLE COMPOUNDS–VI. PHOTOPHYSICAL PROPERTIES OF MONOMERIC MODELS FOR POLYSTYRENE-BOUND PORPHYRINS

Abstract— Several porphyrin esters used as models for polystyrene-bound porphyrins have been prepared and their excited states have been studied by laser flash photolysis, IR phosphorescence of singlet molecular oxygen, O₂(¹Δ_g), and steady-state fluorescence. The photophysical properties of the porphyrin esters in solution are affected by the presence of nitro group(s) in the chain. In this case, an important decrease in φ_f, φ_T and φ_δ (to ca 0.7–0.4 of the value for the parent dimethyl ester) is observed. This is mainly due to intramolecular electron-transfer quenching [by the nitro group(s)] of the first excited singlet state of the porphyrin. The thermodynamic feasibility of this deactivation pathway has been confirmed polarographically. Quenching of the porphyrin triplet state and of O₂(¹Δ_g) by the nitro groups is negligible. The present conclusions explain also the results obtained previously for the photooxidation of bilirubin sensitized by the parent insoluble polystyrene-bound porphyrins. In that case the photooxidation rates were correlated directly with the quantum yield of O₂(¹Δ_g) production by the sensitizer. The consequences of these results for the use of polystyrene-bound porphyrins in sensitized photooxidation processes are discussed.     

THE PHOTOCHEMISTRY OF RIBOFLAVIN—VI. THE PHOTOPHYSICAL PROPERTIES OF ISOALLOXAZINES

Abstract— Many of the photophysical properties of riboflavin and several other N-10 substituted isoal-loxazines have been measured and these include: quantum yields of fluorescence at 77 K and 298 K, φ, quantum yields of phosphorescence at 77 K, φ_P, lifetimes of the triplet state by electron spin resonance and phosphorescence at 77 K, φp,. and the quantum yield of intersystem crossing. φ_isc. For riboflavin in an alcoholic matrix at 77 K the limiting values were: φ _J = 0.32, φ_P= 0.007, φp = 0.20s and φ_isc= 0.7. At 298 K, φ _f for riboflavin in water and alcohol were 0.25 and 0.32, respectively. The results for the photophysical processes are compared with several photochemical processes known to involve the triplet state of riboflavin in aqueous solution. It is concluded that the φ _isc decreases from 0.7 in alcohol to 0.6 in water at 298 K.     

噻唑橙类菁染料与生物大分子结合的光谱特性的研究

研究了两个噻唑橙类菁染料TO-1和TO-2与核酸、蛋白质结合的光谱特性.发现染料喹啉环中氮上取代基对染料标记生物大分子的荧光特性有着很大的影响.羧基的引入,使染料TO-2与核酸结合后的荧光性能下降,但同时也提高了它与蛋白质结合的光物理性能.研究表明:在喹啉环氮上引入适当的亲水性取代基,可以使噻唑橙类菁染料的应用范围拓展到蛋白质标记领域.并初步研究了蛋白质浓度的变化对染料TO-2的荧光光谱的影响以及TO-2在不同pH值的蛋白质溶液中的荧光性质.     

UPTAKE KINETICS AND INTRACELLULAR LOCALIZATION OF HYPOCRELLIN PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY: A CONFOCAL MICROSCOPY STUDY

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.     

PHOTOPHYSICAL PROPERTIES OF NUCLEIC ACID COMPONENTS—1. THE PYRIMIDINES: THYMINE, URACIL, N, N-DIMETHYL DERIVATIVES AND THYMIDINE

Abstract— Low-temperature (and some room temperature) absorption and emission, fluorescence and phosphorescence, data including quantum yields and lifetimes have been obtained from the title pyrimidine bases as a function of the nature of the solvent environment. Modest vibrational resolution has been observed for the first time in the absorption spectra, particularly for thymine and uracil. The excitation spectra also show structure. The quantum yields of fluorescence (φ_F) and phosphorescence are independent of the excitation wavelength. Thymine, thymidine and uracil have profoundly different photophysical properties in polar-aprotic vs polar-protic solvents. The N, N-dimethyl substitution of thymine and uracil produces photophysical changes comparable to the solvent change for the unsubsti-tuted bases. The species involved in the emission processes is the keto (lactam) form. It is probable that ^1,3(n,π*) state(s) has(have) changed order relative to a lowest ¹(π,π*) state as a consequence of both the solvent change and N, N-dimethyl substitution. The lowest triplet state is assigned as ³(n π*). We propose that an important factor contributing to the previously reported excitation wavelength dependence of φ_F and φ_T1 (φ_isc) for nucleic-acid components is the equilibrium coexistence of H-bonded and non-H-bonded forms each having different photophysical properties. Consideration is given of the impact of the significantly different photophysical properties of nucleic-acid bases as a function of the nature of the solvent upon the photochemical properties.     

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.     

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.     

THE USE OF INTERNALIZABLE DERIVATIVES OF CHLORIN E6 FOR INCREASING ITS PHOTOSENSITIZING ACTIVITY

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e₆ conjugates with internalizable ligands as compared to that of chlorin e₆ itself. Receptor-mediated internalization of chlorin e₆ conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.     

PHOTOSENSITIZING PROPERTIES OF BACTERIOCHLOROPHYLLIN a and BACTERIOCHLORIN a, TWO DERIVATIVES OF BACTERIOCHLOROPHYLL a

Abstract The photosensitizing properties of two water soluble derivatives of bacteriochlorophyll a , bacteriochlorophyllin a and bacteriochlorin a (lacking the central Mg-ion) were investigated and compared to those of hematoporphyrin derivatives. At physiological pH the oxygen consumption rate of histidine, tryptophan, dithiothreitol and guanosine upon illumination was 3 to 4 times higher when bacteriochlorin a was used as photosensitizer than when hematoporphyrin derivatives were used. Especially bacteriochlorin a proved to be an effective sensitizer for the killing of L929 cells. Because bacteriochlorin a has an absorption maximum at 765 nm in phosphate buffered saline (allowing a light penetration in tissue about ten times larger than at 630 nm) and a high molar absorption coefficient (32 000 M cm⁻¹) it has promising possibilities for the application in photodynamic therapy.     

CORRELATION BETWEEN THE TRIPLET PHOTOPHYSICAL PROPERTIES AND THE PHOTOBIOLOGICAL ACTION IN YEAST OF TWO MONOFUNCTIONAL PYRIDOPSORALENS

The photophysical properties of the lowest excited triplet states of pyrido[3,4-c]psoralen (PyPs) and 7-methylpyrido[3,4-c]psoralen (MePyPs) have been investigated by laser flash photolysis, including energy transfer from these triplets to oxygen-generating singlet oxygen. A parallel study of the photosensitization of yeast in vivo by these compounds in the presence and absence of oxygen is also reported. The low triplet and hence singlet oxygen yields, reflected in the lack of an oxygen effect in yeast, suggest that photoadditions to DNA are likely to be the main source of the photosensitized lethal effects induced by these pyridopsoralens in vivo.