Robert W. Redmond

Spatially resolved cellular responses to singlet oxygen.

Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 μs in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only ?220 nm during three lifetimes (?5%1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo‐oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 1O2 occur in cells.Abstract Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 μs in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only ∼220 nm during three lifetimes (∼5% 1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo-oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 1O2 occur in cells.

[2] Photosensitized production of singlet oxygen

Photosensitization is a simple and controllable method for the generation of singlet oxygen in solution and in cells. Methods are described for determining the yield of singlet oxygen in solution, for measurement of the rate of reaction between singlet oxygen and a substrate, and for comparing the effectiveness of singlet oxygen generated by different photosensitizers in cells. These quantitative measurements can lead to better understanding of the interaction of singlet oxygen with biomolecules.

DIRECT DETECTION OF SINGLET OXYGEN SENSITIZED BY HAEMATOPORPHYRIN AND RELATED COMPOUNDS

Abstract— Direct time‐resolved detection of the luminescence at 1270 nm from ‘singlet oxygen’ was used to estimate the quantum yield of singlet oxygen production (ΦΔ) from a series of related porphyrins in benzene and in D2O. From this and available data the fraction of oxygen quenching interactions leading to singlet oxygen production (SΔ) was derived in most cases. A marked increase in ΦΔ value was observed for di‐haematoporphyrin ester (DHE) in cetyltrimethyl ammonium bromide/D2O solution in comparison to D2O alone, this increase is attributed to a major structural alteration of DHE on introduction of the detergent.

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD‐MA)*

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD‐MA), a second‐generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet‐state, singlet oxygen (O2(1Δg)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin‐type molecule shows a strong absorbance in the red (λ≈ 688 nm, ɛ≈ 33 000 M−1 cm−1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, Es= 42.1 kcal mol−1, lifetime, Φf= 5.2 ns and fluorescence quantum yield, Φf= 0.05 in air‐saturated solution. The triplet state of BPD‐MA has a lifetime, τf >. 25 ns, an energy level, ET= 26.9 kcal mol−1 and the molar absorption coefficient is ɛT= 26 650 M−1 cm−1 at 720 nm. A dramatic effect of oxygen on the fluorescence (φf) and intersystem crossing (φT) quantum yields has been observed. The BPD‐MA presents rather high triplet (φT= 0.68 under N2‐saturated conditions) and singlet oxygen (φΔ= 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (φPb) of which was found to be on the order of 5 × 10−5 in organic solvents.

The effects of aggregation, protein binding and cellular incorporation on the photophysical properties of benzoporphyrin derivative monoacid ring A (BPDMA)

Absorption, fluorescence and laser flash photolysis spectroscopies were used to investigate the effects of self-aggregation, binding to human serum albumin and incorporation in cancer cells on the photophysics of benzoporphyrin derivative monoacid ring A (BPDMA). Aggregation of BPDMA has been studied in mixtures of methanol and phosphate-buffered saline (PBS). The extent of aggregation was dependent on dye concentration and solvent composition, becoming particularly marked in mixtures containing less than 30% methanol. A dimerization constant Kd or 9 x 10(6) M-1 was determined by fluorescence experiments for BPDMA in pure PBS. In addition to spectral modifications, aggregation induces a lowering of the fluorescence and intersystem crossing quantum yields. Human serum albumin binds BPDMA with an association constant Kb of 5.2 x 10(5) M-1 in PBS. When bound to HSA, BPDMA displays photophysical properties very similar to the monomer in organic solvents. The molar ratio [HSA]/[BPDMA] corresponding to complete binding of the dye was determined to be approximately 10. Efficient generation of the triplet state of BPDMA was also observed from aqueous cellular suspensions containing incorporated photosensitizer.

Time-resolved thermal lensing and phosphorescence studies on photosensitized singlet molecular oxygen formation. Influence of the electronic configuration of the sensitizer on sensitization efficiency

Abstract The absolute quantum yields, Φ Δ , of photosensitized formation of singlet molecular oxygen, O 2 ( 1 Δ g ), by various sensitizers in benzene were measured by time-resolved thermal lensing (TRTL). Relative measurements with time-resolved O 2 ( 1 Δ g ) phosphorescence detection were used to confirm the absolute values from TRTL. For aromatic ππ* triplet states, S Δ = Φ Δ /Φ ISC = 0.9 ± 0.1 and the rate constant for quenching of the triplet state of O 2 , k q = (2 ± 0.5 ) × 10 −1 M 9 s −1 , is about 1/9 of the diffusional rate constant. For the more polarizable nπ* triplet states S Δ = 0.3 ± 0.05 and k q sharply increases with triplet energy.

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.

Collagen Cross-Linking Using Rose Bengal and Green Light to Increase Corneal Stiffness

PURPOSE Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application. METHODS Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment. RESULTS RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability. CONCLUSIONS RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

EFFICIENCY OF THE PHOTOPROCESSES LEADING TO SINGLET OXYGEN (1δg) GENERATION BY α‐TERTHIENYL: OPTICAL ABSORPTION, OPTOACOUSTIC CALORIMETRY AND INFRARED LUMINESCENCE STUDIES*

Abstract— …The triplet energy of α‐terthienyl has been determined by heavy atom‐induced optical absorption; the value of 39.7 ±1.5 kcal/mol is consistent with earlier energy transfer work. Combining this result with calorimetric data from optoacoustic calorimetry indicates that intersystem crossing occurs with at least 90% efficiency in polar and non‐polar solvents. The quantum yields for singlet oxygen formation via energy transfer from triplet α‐terthienyl have been obtained from time‐resolved measurements of its IR phosphorescence; these yields are in the 0.6‐0.8 range in non‐polar and polar (hydroxylic and non‐hydroxylic) solvents.

Photophysical properties of 3,3'-dialkylthiacarbocyanine dyes in homogeneous solution.

The photophysical properties of 3,3′‐dialkylthiacarbocyanine iodides and chlorides were measured in various solvents. It was found that photoisomerization and fluorescence are the major contributors to the deactivation of the excited singlet state; intersystem crossing occurs with only a very low efficiency. In ethanol, a triplet yield of 0.004 and a singlet oxygen quantum yield of 0.002 were determined. The photophysical parameters of these dyes are not substantially influenced by the length of the alkyl chain or the size of the halide counterion. The substitution of an ethyl with an octadecyl‐chain only slightly hinders photoisomerization, and the replacement of the chloride with an iodide reduces only marginally the fluorescence lifetimes and fluorescence quantum yields in chloroform. A significant external heavy‐atom effect is observed using dibromoethane as a solvent: triplet and singlet oxygen yields increase7–10‐fold, and the triplet lifetime decreases from 55 μs to 15 mUs.