Review of photodynamic therapy for gastrointestinal tumours in the past 6 years in China

Patients (142) with advanced gastrointestinal (GI) cancers were treated by photodynamic therapy (PDT) from September 1982 to December 1988. Haematoporphyrin derivative (HpD) (5 mg kg-1) was intravenously given 48-72 h prior to PDT. The light source was an argon dye laser emitting at 630 nm. The entire tumour was irradiated with a light dose of 100-250 J cm-2. Fifteen patients (10.6%) showed complete response (CR), 53 (37.3%) showed partial response (PR) and 32 (22.5%) showed mild response (MR). The clinical results show that the estimated energy dose (EED) of 200-250 J cm-2 is appropriate. All patients were treated by PDT and adjuvant chemotherapy showing good results with a follow-up of 1-5 years; 13 out of 15 patients in the CR group are alive (86.7%); 12 patients survived more than 2 years (8.8%).     

ACTION SPECTRUM (620–640 nm) FOR HEMATOPORPHYRIN DERIVATIVE INDUCED CELL KILLING

Abstract— Monochromatic red light generated by a tunable dye laser is currently being utilized for the treatment of solid tumors with hematoporphyrin derivative (HpD) photoradiation therapy (PRT). Experiments were performed using mammalian cells to determine the most efficient wavelength of red light (620 to 640 nm range) for HpD induced cellular photoinactivation. Decrease in the clonogenic potential of Chinese hamster ovary (CHO) cells was examined following both short (I h) and extended (12 h) HpD incubation times. Maximal photosensitization was observed with wavelengths ranging from 630 to 632.5 nm and the action spectra for cell killing matched the absorption spectra for HpD bound to cells. Similar observations were obtained following both short and extended HpD-cell incubation times. The potential relevance of these results as they relate to clinical HpD PRT are discussed.     

Lasers used in analytical micropyrolysis

Laser micropyrolysis gas chromatography mass spectrometry (GC-MS) allows analytical pyrolysis to be conducted with micro-spatial resolution. Despite the large range of contemporary laser sources, most previous laser pyrolysis studies have been conducted with continuous wave (CW) infrared irradiation. Here, the laser micropyrolysis analysis of a Sydney torbanite was conducted with three different laser sources - 1. CW 532 nm; 2. Q-Switched (QSw) pulsed 1064 nm; and 3. QSw pulsed 266 nm - to compare the molecular analyses attributes of different laser types (λ: 266-1064 nm; CW or QSw). The CW 532 nm laser irradiation consistently produced high concentrations of n-hydrocarbons, with lesser amounts of cyclic and aromatic hydrocarbons, similar to previous analyses with both CW 1064 nm laser pyrolysis and conventional analytical pyrolysis [1]. In contrast, both the IR and UV QSw pulsed irradiation sources provided poor and varied data. Relatively low concentrations of n-hydrocarbons were occasionally produced, but most often no structurally significant products were detected. The poor maintenance of hydrocarbon structural units by the short pulse lasers can be attributed to the very high power density delivered, leading to excessive degradation of the irradiated macromolecule.     

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effects of visible femtosecond laser pulses and continuous wave laser radiation of low average intensity on the clonogenicity of Escherichia coli.

To evaluate the contribution of local pulsed heating of light-absorbing microregions to biochemical activity, irradiation of Escherichia coli was carried out using femtosecond laser pulses (λ = 620 nm, τ_p=3 × 10⁻¹³ s, f_p = 0.5 Hz, E_p = 1.1 × 10⁻³J cm⁻², I_av = 5.5 × 10⁻⁴ W cm⁻², I_p = 10⁹ W cm⁻²) and continuous wave (CW) laser radiation (λ = 632.8 nm, I = 1.3 W cm⁻²). The irradiation dose required to produce a similar biological effect (a 160%–190% increase in the clonogenic activity of the irradiated cells compared with the non-irradiated controls) is a factor of about 10³ lower for pulsed radiation than for CW radiation (3.3 × 10⁻¹ and 7.8 × 10² J cm⁻² respectively). The minimum size of the microregions transiently heated on irradiation with femtosecond laser pulses is estimated to be about 10 Å, which corresponds to the size of the chromophores of hypothetical primary photoacceptors—respiratory chain components.     

Control of photobleaching in photodynamic therapy using the photodecarbonylation reaction of ruthenium phthalocyanine complexes via stepwise two-photon excitation

In this study, we have investigated the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes. When a nanosecond-pulsed laser is used, the photodecarbonylation of our Ru complexes efficiently proceeds via stepwise two-photon excitation, while the reaction yields are negligibly small when a continuous-wave (CW) laser is employed. The pulsed laser selective photodecarbonylation decreases the Q-band absorbance, which satisfies what the photodynamic therapy (PDT) requires of the photobleaching. For RuPc(CO)(Py), the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro. Toxicity and phototoxicity tests indicate that our RuPc(CO)(Py) and RuNc(CO)(Py) complexes in concentrations of 0.3-1 microM and 1-2 microM, respectively, are applicable as PDT agents. The phototoxicity is consistent with the photochemical properties of these complexes, namely, excited triplet lifetimes (10 and 4.8 micros for the Pc and Nc complexes, respectively) and singlet oxygen yields (0.48 and 0.35 for the Pc and Nc complexes, respectively). On the basis of these results, we propose a novel concept for achieving a greater depth of necrosis in PDT as follows: (1) PDT of upper cellular layers using CW-laser irradiation; (2) efficient photobleaching in upper cellular layers using pulsed dye-laser irradiation, which results in an increase in the therapeutic depth of red light; (3) PDT directed toward deeper tumor tissues using CW laser irradiation. In addition, these Ru complexes are promising as CO release agents for investigative biochemistry.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

LOCALIZATION OF MONO-L-ASPARTYL CHLORIN e6 (NPe6) IN MOUSE TISSUES

Abstract It is known that HpD is retained longer by malignant tissue than normal tissue and is therefore a useful material for photodynamic therapy (PDT). Currently, vigorous research is being conducted throughout the world to discover a new material which can have greater cancer cell affinity than hematoporphyrin derivative (HpD) and will be used effectively for PDT. Investigation has been conducted to determine the spectral characteristics and cancer cell affinity of NPe6, a recently developed material.
Structurally, a double bond on the D-ring of the porphyrin ring of mono-L-aspartyl chlorin e6 (NPe6) has been reduced, thereby changing its spectral properties from that of HpD. This difference accounts for the stronger absorption bands in wavelengths longer than those of HpD. Furthermore, NPe6 in tumor showed stronger absorption at 660 nm than HpD. Absorption by hemoglobin (Hb) in the blood occurs at wavelengths in the range 500-600 nm, thereby lowering light transmittance. A compound which has a strong absorption band at wavelengths longer than 600 nm and consequently is not affected by Hb will naturally be activated by light at a greater depth in tissue than compounds which do not share this characteristic. The localization of NPe6 in sarcoma and various internal organs was examined with an endoscopic spectrophotometer using an excimer dye laser. After 72 h i.v. NPe6 injection, the results indicate that NPe6 has 10 times greater uptake in malignant tissue cells than in normal organs. Based on the above observations, it was concluded that NPe6 could be effective for PDT if toxicity is low and that this compound has a high malignant tissue affinity.     

Photosensitizers in dermatology

Systemic injection of hematoporphyrin derivative (HpD) in contribution with visible light (red or blue-green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea-pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.     

WAVELENGTH DEPENDENCE OF HEMATOPORPHYRIN DERIVATIVE PHOTODYNAMIC TREATMENT EFFECTS ON RAT EARS

The in vivo wavelength dependence of hematoporphyrin derivative (HpD) photodynamic treatment (PDT) has been studied. Ears of 136 rats were treated at six red and four blue-green laser wavelengths (615-635, 488-514.5 nm). Hematoporphyrin derivative was administered intraperitoneally (15 mg/kg) and 24 h later both ears were irradiated, at different wavelengths, for t = 6.5, 10 or 15 min at 60 mW/cm2. Four parameters (thickness, average erythema, eschar and loss of tissue) were quantified and a combined score (CS) of effects was established statistically. The maximum combined score during follow-up was taken as a measure for the biological effect. The light distribution in rat ears during irradiation with red and blue-green light was estimated from in vivo measurements and the transport theory. Statistical analysis of the combined score data yielded values for the relative biological effectiveness (RBE). Relative biological effectiveness maxima occurred at 501.7 and 625 nm. Analyzing erythema and loss of tissue separately yielded maxima at the same wavelengths. Quantitative agreement between the latter two sets of relative biological effectiveness values was obtained only when they were referred to the actual light energy fluence in tissue, rather than to the incident fluence. These relative biological effectiveness values are about 2.3 at 501.7 nm and 1.35 at 625 nm, taking relative biological effectiveness = 1 at 630 nm.     

Irradiation of Amelanotic Melanoma Cells with 532 nm High Peak Power Pulsed Laser Radiation in the Presence of the Photothermal Sensitizer Cu(II)-Hematoporphyrin: A New Approach to Cell Photoinactivation

Cu(II)-hematoporphyrin (CuHp) was efficiently accumulated by B78H1 amelanotic melanoma cells upon incubation with porphyrin concentrations up to 52 microM. When the cells incubated for 18 h with 13 microM CuHp were irradiated with 532 nm light from a Q-switched Nd: YAG laser operated in a pulsed mode (10 ns pulses, 10 Hz) a significant decrease in cell survival was observed. The cell photoinactivation was not the consequence of a photodynamic process, as CuHp gave no detectable triplet signal upon laser flash photolysis excitation and no decrease in cell survival was observed upon continuous wave irradiation. Thus, it is likely that CuHp sensitization takes place by photothermal pathways. The efficiency of the photoprocess was modulated by different parameters; thus, while varying the amount of added CuHp in the 3.25-26 microM range had little effect, pulse energies larger than 50 mJ and irradiation times of at least 120 s were necessary to induce a cell inactivation of about 50%. The porphyrin-cell incubation time prior to irradiation had a major influence on cell survival, suggesting that the nature of the CuHp microenvironment can control the efficiency of photothermal sensitization.     

MEMBRANE LYSIS IN CHINESE HAMSTER OVARY CELLS TREATED WITH HEMATOPORPHYRIN DERIVATIVE PLUS LIGHT

Abstract Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [³H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.     

Effect of light on calcium transport in bull sperm cells.

The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Ca2+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca2+ transport in the irradiated cells, which means that laser light can stimulate Ca2+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.     

GIANT CELL FORMATION IN BLADDER TUMOR CELLS FOLLOWING HEMATOPORPHYRIN DERIVATIVE-SENSITIZED PHOTOIRRADIATION

Abstract— Giant cell induction was studied in cultured bladder tumor cells (MGH-Ul; commonly referred to as "EJ") following hematoporphyrin derivative (HpD)-sensitized photoirradiation. Eight days after treatment, control colonies (no treatment, HpD alone, light alone) were generally uniform in size and density and contained few giant cells. Colony heterogeneity and giant cell frequency increased with light dose in the presence of HpD. The morphology and frequency of giant cell formation, with respect to survival levels, were comparable to those observed in 280 kVp X-irradiation of the same cells.     

Two-photon photodynamic therapy of C6 cells by means of 5-aminolevulinic acid induced protoporphyrin IX

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4-5h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 x 10(10) W/cm(2), while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 x 10(10) W/cm(2). The observed affected area of the cell layers was between 0.13 mm(2) and 1.10 mm(2). Since the irradiation of non-incubated cells below the mean power density of 10.9 x 10(10) W/cm(2) induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.     

Use of photodynamic therapy in the treatment of vocal cord carcinoma

The photodynamic therapy of superficial vocal cord carcinoma is a promising new technique. Patients were irradiated with 630 nm light from an Aurora dye laser under general anaesthesia 72 h after a haematoporphyrin derivative (HpD) injection. So far, 32 patients have been treated for small epidermoid carcinoma of the glottis without anterior commissure. There have been no undesired effects due to the photosensitizer or the dye laser. The results show seven failures and 25 positive responses, of which three patients were treated more than 4 years ago.     

Generation and interrogation of a pure nuclear spin state by parahydrogen-enhanced NMR spectroscopy: a defined initial state for quantum computation

We describe a number of studies used to establish that parahydrogen can be used to prepare a two-spin system in a pure state, which is suitable for implementing NMR quantum computation. States are generated by pulsed and continuous-wave (CW) UV laser initiation of a chemical reaction between Ru(CO)(3)(L(2)) [where L(2) = dppe = 1,2-bis(diphenylphosphino)ethane or L(2) = dpae = 1,2-bis(diphenylarsino)ethane] with pure parahydrogen (generated at 18 K). This process forms Ru(CO)(2)(dppe)(H)(2) and Ru(CO)(2)(dpae)(H)(2) on a sub-microsecond time-scale. With the pulsed laser, the spin state of the hydride nuclei in Ru(CO)(2)(dppe)(H)(2) has a purity of 89.8 +/- 2.6% (from 12 measurements). To achieve comparable results by cooling would require a temperature of 6.6 mK, which is unmanageable in the liquid state, or an impractical magnetic field of 0.44 MT at room temperature. In the case of CW initiation, reduced state purities are observed due to natural signal relaxation even when a spin-lock is used to prevent dephasing. When Ru(CO)(3)(dpae) and pulsed laser excitation are utilized, the corresponding dihydride product spin state purity was determined as 106 +/- 4% of the theoretical maximum. In other words, the state prepared using Ru(CO)(3)(dpae) as the precursor is indistinguishable from a pure state.     

Hypericum perforatum L. extract - novel photosensitizer against human bladder cancer cells

The polar methanolic fraction (PMF) of the Hypericum perforatum L. extract has recently been developed and tested as a novel, natural photosensitizer for use in the photodynamic therapy (PDT), and photodynamic diagnosis (PDD). PMF has been tested on HL-60 leukemic cells and cord blood hemopoietic progenitors. In the present study, the efficacy of PMF as a phototoxic agent against urinary bladder carcinoma has been studied using the T24 (high grade metastatic cancer), and RT4 (primary low grade papillary transitional cell carcinoma) human bladder cancer cells. Following cell culture incubation, PMF was excited using 630 nm laser light. The photosensitizer exhibited significant photocytotoxicity in both cell lines at a concentration of 60microg/ml, with 4-8 J/cm(2) light dose, resulting in cell destruction from 80% to 86%. At the concentration of 20microg/ml PMF was not active in either cell line. These results were compared with the results obtained in the same cell lines, under the same conditions with a clinically approved photosensitizer, Photofrin. Photofrin was used in the maximum clinically tolerable dose of 4microg/ml, and it was also excited with 630 nm laser light. In the T24 cell Photofrin exhibited slightly less photocytotocixity, compared with PMF, resulting in 77% cell death with 8J/cm(2) light dose. However, against the RT4 cells Photofrin resulted in minimal cell death (9%) with even 8J/cm(2) light dose. Finally, the type of cell death induced by PMF photoactivation was studied using flow cytometry and DNA laddering. Cell death by PMF photodynamic action in these two bladder cell lines is caused predominently by apoptosis. The reported significant photocytotoxicity, selective localization, natural abundance, easy, and inexpensive preparation, underscore that the PMF extract hold the promise of being a novel, effective PDT photosensitizer.     

1064nm激光诱导Ge(111)与Cl2反应动力学

采用超声分子束和时间分辨质谱技术研究了1064nm脉冲激光辐照下Ge(111)与Cl₂的反应动力学。实验结果表明,该反应的主要产物为GeCl₂,提高入射氯分子的平动能将增加反应速率。激光能量密度对GeCl₂产率呈指数关系,而对GeCl₂的平动温度影响不大。升高Ge(111)表面温度也能提高反应产率。同时还讨论了近红外激光诱导GeCl₂反应的机理。     

Mist separation and sonochemiluminescence under pulsed ultrasound

Differences in the amount of water-mist separation and the intensity of luminol chemiluminescence for pulsed and continuous-wave (CW) ultrasound at 135 kHz have been investigated. The amount of mist generated is estimated using the cooling rate of a copper plate sprayed with the mist. For pulsed operation with an appropriate duty cycle, the cooling rate and the cooling rate per input power to the transducer are higher by 4 and 12 times compared to CW operation, respectively. This is due to the amplitude of the pulsed ultrasound being higher than that for CW ultrasound. Relatively low power pulsed operation can successfully produce both a higher sonochemiluminescence (SCL) intensity and cooling rate than those for CW ultrasound. The sonochemical reaction for pulsed ultrasound occurs at the same input power threshold as that for mist separation, whereas for CW ultrasound, the former threshold is lower than the latter. A higher number of large bubbles is produced with CW ultrasound than that with pulsed ultrasound. To achieve a sound pressure amplitude sufficient for mist separation near the surface of a liquid, it is necessary to expel these bubbles by changing the sound field from resonant standing waves to progressive waves that give rise to capillary waves on the liquid surface.