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To enhance the potency of photosensitizer, we developed a novel photosensitizer, Laserphyrin®-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin®, Meiji Seika Pharma) into hemagglutinating virus of Japan envelope (HVJ-E). In this study, we examined the optimal Laserphyrin® concentration for preparation of Laserphyrin®-HVJ-E which had photocytotoxicity and maintained direct cytotoxicity derived from HVJ-E. Then, potency of Laserphyrin®-HVJ-E and Laserphyrin® were compared in vitro using castration-resistant prostate cancer cell line (PC-3). A laser diode (L660P120, Thorlabs, USA) with a wavelength of 664 nm was used for light activation of Laserphyrin®, which corresponds to an absorption peak of Laserphyrin® and provides a high therapeutic efficiency. The photocytotoxicity and direct cytotoxicity of Laserphyrin®-HVJ-E prepared using various Laserphyrin® concentrations were evaluated using PC-3 cell in vitro. We categorized the treatment groups as Group 1: 50 μL of D-MEM treatment group, Group 2: HVJ-E treatment group, Group 3: Laserphyrin®-HVJ-E treatment group, and Group 4: Laserphyrin® treatment group. Group 3 was subjected to different concentrations of Laserphyrin®-HVJ-E suspension, and all groups were subjected to different incubation periods (24, 48 h), (30 min, 1 h, or 3 h,) respectively, without and after PDT. Laserphyrin®-HVJ-E prepared using 15 mM Laserphyrin® had high photocytotoxicity and maintained HVJ-E’s ability to induce direct cytotoxicity. Therapeutic effect of Laserphyrin®-HVJ-E was substantially equivalent to that of Laserphyrin® alone even at half Laserphyrin® concentration. By utilizing Laserphyrin®-HVJ-E, PDT could be performed with lower Laserphyrin® concentration. In addition, Laserphyrin®-HVJ-E showed higher potency than Laserphyrin® by combining cytotoxicities of HVJ-E and PDT.

In photodynamic therapy (PDT), singlet oxygen ([Formula: see text]) is the main species responsible for promoting tumor cell death. The determination of the quantum yield (ΦΔ) of a photosensitizer (PS) is important for dosimetry. The purpose of this paper is to quantify the [Formula: see text] generated by the PS by near-infrared spectroscopy (NIRS). The ΦΔ of different PS species were measured by the detection of near-infrared [Formula: see text] luminescence. From the measurement results, the ΦΔ of talaporfin sodium, protoporphyrin IX (PpIX), and lipidated PpIX (PpIX lipid) were measured as 0.53, 0.77, and 0.87, respectively. In addition, the ΦΔ values of PpIX in a hypoxic and oxic solution were evaluated, since tumors are associated with regions of hypoxia. The measured ΦΔ indicated a same value at high (DO: 20%) and low (DO: 1%) oxygen concentrations. Using the measured ΦΔ, the amount of [Formula: see text] generated by the PSs was estimated using [[Formula: see text]] = D*ΦΔ, where D* is the total excited PS concentration. The generated [Formula: see text] amounts were little different at the high and the low oxygen concentrations, and the generated [Formula: see text] amount for each PS was different depending on each ΦΔ. The NIRS measurement determined the ΦΔ of talaporfin sodium, PpIX, and PpIX lipid. The quantitative evaluation based on the measured ΦΔ will support the development of PDT treatment monitoring and design.

Endoscopic submucosal dissection (ESD) has been at the forefront of international attention as a less invasive treatment for early gastrointestinal cancer. Currently, ESD involves the use of an electrosurgical knife for mucosal incision and subsequent submucosal dissection. However, it has been reported that perforation occurs in approximately 5% of cases. To enhance tissue selectivity with this modality, we focused on applying a laser to ESD (laser ESD). A CO2 laser was chosen as the surgical knife because the saline or sodium hyaluronate solution injected into the submucosal layer during the current ESD procedure has a high absorption coefficient at the wavelength of the CO2 laser. Thus, the purpose of this study is to quantitatively clarify the safety and efficacy of laser ESD for the colon. First, we validated a porcine colon as a model of the human colon in terms of optical and thermal properties. Next, ex vivo experiments on the safety and efficacy of laser ESD were performed. In ex vivo experiments using extracted porcine colon tissue, an incision depth of 0.5–1.0 mm was obtained without thermal damage to the muscle layer when the power density was set at 17, 22, or 28 W/mm2. In addition, less thermal damage was observed in tissue incised with this method compared with electrosurgical knives. These results might be explained by the strong absorption of the CO2 laser by the saline injected into the submucosa. Therefore, laser ESD is expected to be a safer method for the treatment of early colon cancer.

Photobleaching and photoproduct formations are considered essential phenomena in improving the efficacy of photodynamic diagnosis and therapy (PDD and PDT). We investigated the photobleaching of protoporphyrin IX (PpIX) by measuring its concentration with mass spectrometry (MS). The reduction in the concentration of PpIX dissolved in dimethyl sulfoxide was measured during PDD and PDT conditions using lasers with wavelengths of 405 and 635 nm, respectively, at a power density of 10, 50, or 100 mW/cm2 . The obtained results were compared with the results of conventional fluorescence spectroscopy and previously reported results. Our results demonstrate the variation in the MS-based photobleaching coefficient of PpIX with the power density, while the fluorescence-based photobleaching coefficient was independent of the power density. The results of MS also show faster photobleaching of PpIX in comparison with that obtained from fluorescence. The difference may be attributed to the change in the fluorescence quantum yield of PpIX with its concentration and the effect of fluorescence emission from the PpIX photoproducts. Thus, an MS-based investigation of the photobleaching poses to be a more stable investigation form. Our finding highlights the importance of recognizing the potential significance of these discoveries in the PDD and PDT dosimetry and efficacy.

Photodynamic diagnosis (PDD) of gastric cancer with protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) will lead to a method to estimate the invasive depth. The excitation light for ALA-based PDD is attenuated by absorption and scattering of the mucosal tissue according to its wavelength and the difference contains the fluorophore depth information. To realize the method to estimate the invasive depth for the intramucosal cancer, this study investigated the differences of the attenuation rate between two excitation wavelengths. Considering the optical properties of mucosa and PpIX, relationships between the invasive depths of cancer containing PpIX and the fluorescence intensity ratios detected from the mucosa were estimated using a numerical model. An experiment was also performed using a pellet made of epoxy resin containing PpIX. The excitation lights with wavelengths of 405 and 505 nm having the same power density were each used to irradiate the pellet placed at the fluorophore depth of 0 mm, 1.0 mm, and 1.7 mm from the mucosal surface to obtain fluorescence images, and the average fluorescence intensities of the images were evaluated. The numerical and experimental results suggested the possibility to estimate the fluorophore depth of the PpIX fluorescence source by comparing the fluorescence images with excitation lights at wavelengths of 405 and 505 nm.

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

Background Subcellular localization of a photosensitizer is known to determine the therapeutic efficacy of photodynamic therapy (PDT). Cell membrane is an optimal target that promises an effective treatment outcome. Objectives We previously developed a novel photosensitizer named porphyrus envelope (PE) by combining hemagglutinating virus of Japan envelope (HVJ-E) with lipidated protoporphyrin IX (PpIX lipid). In the current study, the cellular localization of PE and its ability to induce multiple anti-tumor effect were characterized. Materials and Methods The localization and uptake of PpIX lipid in cells were evaluated with confocal laser scanning microscopy and a cell-based fluorescent assay, respectively. The ability of PE to suppress the migration and proliferation of cancer cells was assessed using a scratch-wound assay. The synergistic effect of PDT and HVJ-E treatment was evaluated using an in vitro experiment with PC-3 cells. Results PE localized along the cell membrane and PpIX lipid accumulated selectively in the prostate cancer cells within 10 min. Also, PE maintained the ability to undergo fusion and induce cancer cell death even after light irradiation at the dose for PDT. Incubation with PE resulted in delayed migratory and proliferative activity of PC-3 cells. PE-mediated PDT was twice as effective when cells were further incubated with PE following PDT. Conclusions PE allows rapid drug delivery targeting the cell membrane. Because the cytotoxicity of HVJ-E was maintained, synergistic effect of HVJ-E and the photochemical reactions resulted in highly effective killing of prostate cancer cells in vitro and thus represents a promising treatment for prostate cancer.

RATIONALE Continuous flow ionization methods using infrared (IR) lasers have several favorable characteristics, including ionization without any additional matrices and tolerance to contaminants such as detergents and buffer salts. However, poor sensitivity due to low ion-transfer efficiency from the sample plate to the inlet capillary of the mass spectrometer under atmospheric pressure remains a serious problem. METHODS We developed a new continuous flow IR laser desorption/ionization (IR-LDI) method using a frit plate and wavelength tunable mid-infrared laser with an optical parametric oscillator. Continuous flow samples were directly injected into the ion source without any additional matrices. The ion source was covered with a decompression chamber, and could vary the pressure of the ion source from 21 to 101 kPa. RESULTS Reduction of the pressure of the IR-LDI source from 101 to 71 kPa increased the signal intensity for the [M+H]+ ion of angiotensin II by 1.8-fold. On the other hand, the ion signal intensity was reduced at pressures lower than 71 kPa. It became clear that reducing pressure was more effective when ionization occurred with lower laser pulse energy and lower ion source temperature. In addition, signal intensities for the [M+2H]2+ and [M+3H]3+ ions of insulin were also increased, by 1.4-fold and 1.1-fold respectively, upon reduction of the pressure to 91 and 81 kPa. CONCLUSIONS Although many studies have described IR-LDI using a differential pumping mass spectrometer, the optimal pressure of the ion source has never been investigated. We found that a slight reduction of pressure enhances sensitivity. This knowledge may be applicable to a number of ambient ionization methods using IR lasers.

Interstitial photodynamic therapy (iPDT) with 5-aminolevulinic acid (ALA) is a possible alternative treatment for malignant brain tumors. Further evaluation is, however, required before it can be clinically applied. Computational simulation of the photophysical process in ALA-iPDT can offer a quantitative tool for understanding treatment outcomes, which depend on various variables related to clinical treatment conditions. We propose a clinical simulation method of ALA-iPDT for malignant brain tumors using a singlet oxygen (O12) model and O12 threshold to induce cell death. In this method, the amount of O12 generated is calculated using a photosensitizer photobleaching coefficient and O12 quantum yield, which have been measured in several previous studies. Results of the simulation using clinical magnetic resonance imaging data show the need to specify the insertion positions of cylindrical light diffusers and the level of light fluence. Detailed analysis with a numerical brain tumor model demonstrates that ALA-iPDT treatment outcomes depend on combinations of photobleaching and threshold values. These results indicate that individual medical procedures, including pretreatment planning and treatment monitoring, will greatly benefit from simulation of ALA-iPDT outcomes.

For safe and effective interstitial PDT (iPDT), treatment planning is required since the efficacy depends on optical irradiation conditions including the number of inserted optical diffusers, the insertion positions, their powers, and so on. We are developing a treatment planning method based on a numerical model of singlet oxygen generation during light irradiation for iPDT. In the model, photosensitizer photobleaching, which occurs usually during light irradiation for iPDT, is taken into consideration. In this paper, we implement a numerical simulation of the efficacy of iPDT of malignant brain tumor with 5ALA according to desired optical irradiation conditions and evaluate quantitatively the photobleaching effect on the efficacy simulation. In the simulation, to calculate light propagation into biotissues, a 3D brain model segmented into tissue types, brain normal and tumor tissues, was constructed using an MRI data of a patient with a brain tumor (tumor volume: 34 cm3). Supposing that 8 optical diffusers (light power density of the diffuser surface: 580 mW/cm2, length: 40 mm, diameter: 1.1 mm) were inserted into the tumor region, the light distribution was calculated. Then, the photobleached ALA and the accumulated singlet oxygen distribution were calculated to obtain the treated volumes depending on singlet-oxygen concentration threshold values to induce cell death. Compared to the estimated treatment volume without the photobleaching effect, the estimated efficacy with the proposed model was decreased. This result indicates that the model with photobleaching effect will lead to improvement of the efficacy prediction of iPDT.

BACKGROUND: Photodynamic therapy (PDT), a minimally invasive cancer treatment involving the activation of photosensitizer by a specific wavelength of light, is considered to be a promising treatment option for drug-resistant prostate cancer. Hemagglutinating virus of Japan envelope (HVJ-E) has the potential to serve as a highly effective cancer therapy through selective drug delivery and enhancement of the anti-tumor immune response. OBJECTIVES: To improve therapeutic efficacy and selective accumulation of photosensitizer into tumor cells, we developed a novel photosensitizer, Laserphyrin(®)-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin(®), Meiji Seika Pharma) into HVJ-E. MATERIALS AND METHODS: The therapeutic effect of PDT with Laserphyrin(®) or L-HVJ-E was evaluated in the human prostate cancer cell line PC-3 in vitro. The subcellular localizations of Laserphyrin(®) and L-HVJ-E were observed by confocal microscopy. Apoptosis or necrosis following PDT was detected by annexin V-fluorescein/propidium iodide double staining. RESULTS: The cytotoxic effect of Laserphyrin(®)- and L-HVJ-E-mediated PDT were determined by evaluating cell survival rate and production of reactive oxygen species. The cytotoxicity of L-HVJ-E-mediated PDT was dependent on drug concentration and light dose. Laserphyrin(®) and L-HVJ-E gradually entered cells as incubation time increased, and both agents tended to be distributed in lysosomes rather than mitochondria. Time and dose dependent increase in ROS production was observed, and induction of both apoptotic and necrotic cell death was confirmed. CONCLUSIONS: Laserphyrin(®) and L-HVJ-E were distributed mainly in lysosomes and induced cell death by both apoptosis and necrosis. Furthermore, L-HVJ-E-mediated PDT effectively killed cultured PC-3 cells and exerted higher photocytotoxicity than Laserphyrin(®)-mediated PDT.

Combined therapy using photodynamic therapy (PDT) and chemotherapy has been proposed for anticancer-drug-resistant cancer cells. To evaluate the efficacy of such a combined therapy, the uptakes of an anticancer drug and a photosensitizer in cancer cells must be assessed. Mass spectrometry using matrix-assisted laser desorption/ionization can detect multiple drugs simultaneously. Human prostate cancer cells PC-3 or docetaxel-resistant cancer cells PC-3-DR were incubated in a serum-free medium containing a photosensitizer, protoporphyrin IX (PpIX), and an anticancer drug, docetaxel. A zeolite matrix was created by mixing 6-aza-2-thiothymine and NaY5.6 zeolite, and dissolving in water with 50% acetone. Ions were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a wavelength of 355 nm. The cell morphology was preserved by washing the cells with ammonium acetate and drying in a vacuum after drug administration. Protonated PpIX (m/z 563.3) and the sodium adduct ion of docetaxel (m/z 829.9) were obtained from PC-3 cells simultaneously using the zeolite matrix. On the other hand, PpIX was detected but ions originating from docetaxel were not detected from PC-3-DR cells. The result indicated the efficacy of PDT for docetaxel-resistant cancer cells.

The protoporphyron (PPIX)-lipid (PL-C17) liposomes were successfully prepared from the corresponding micelles by post-inserted method. Both the PL-C17 micelles and liposomes were distributed in plasma membrane and cytoplasm after incubation of the cells with PL-C17 liposomes for 1h. They translocated from plasma membrane into a certain organelle in the cells after incubation in the photosensitizer-free medium. Higher photo-cytotoxicity was observed in the PL-C17 micelles and liposomes localized in plasma membrane in comparison with those localized in the cytoplasm under light irradiation. The LDH assay revealed that cytopathic damages of the plasma membrane were observed in the PL-C17 micelles and liposomes highly localized in plasma membrane. The fluorescent intensity of the calcein-encapsulating DOPC liposomes post-inserted with PL-C17 increased after light irradiation, suggesting that the membrane disruption is possibly caused by oxidation of membrane lipids with ROS generated from photosensitizers and affects the photo-cytotoxicity in PDT.

Significance: The diagnostic depth of photodynamic diagnosis (PDD) for gastric cancer with protoporphyrin IX (PpIX) is limited, which leads to missing intramucosal cancers in screening and surgery. Aim: The reason is that the excitation light, whose wavelength is determined by the highest absorption peak of PpIX (∼405 nm), is strongly attenuated by mucosal tissues. We investigated an excitation wavelength that can extend the diagnostic depth of PpIX fluorescence at the mucosal subsurface. Approach: By calculating the depth-dependent intensity of the excitation light in porcine gastric mucosa for each wavelength, relationships among the wavelength, fluorophore depth, and fluorescence intensity were assessed and fluorescence images of PpIX pellets located at different fluorophore depths were compared experimentally by changing the excitation wavelength. Results: The numerical calculation showed that a 505-nm excitation light provided the highest fluorescence intensities at a fluorophore depth deeper than 1.1 mm. In the fluorescence observation, the fluorescence intensities at fluorophore depths of 0 and 1.0 mm at 405 nm were 5.4 × 103 and 1.0 × 103 arb. units, whereas those at 505 nm were 5.3 × 101 and 1.9 × 102 arb. units, respectively. Conclusion: The experimental results suggest that the diagnosis depth of PDD with PpIX for intramucosal cancer can be extended by 505-nm excitation light.

Since the increase in the overall mortality rate in patients with colon cancer is remarkably high in recent years, early treatment is required. For this reason, endoscopic submucosal dissection (ESD) has been at the forefront of international attention as a low invasive treatment for early digestive cancer. In current ESD procedure, an electrosurgical knife is used for mucosal incision and subsequent submucosal dissection. However, the perforation has been reported to occur by approximately 5%. Thus, to enhance the tissue selectivity of this modality, we focused on the application of laser for ESD. A carbon dioxide laser was chosen as a surgical knife because the saline or a sodium hyaluronate solution injected into the submucosal layer in current ESD procedure has a high absorption coefficient at the wavelength of the carbon dioxide laser. In this research, ex vivo experiment was performed at the output power of 3–7 W and discuss the optimum irradiation power of laser. As a result of ex vivo experiment using extracted porcine colon tissues, mucosal incision and submucosal dissection were safely and less invasively performed in every output power, without reaching the thermal damage to a muscular layer. This is because a carbon dioxide laser is strongly absorbed by saline injected into submucosa. ESD using a carbon dioxide laser is a safer method for the treatment of early colon cancer. We are planning to measure and compare the optical and thermal properties of porcine colon with those of human colon.

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix-assisted laser desorption/ionization (IR-MALDI). Especially in the 6–7 µm-band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O–H, CO, and CH3. Additionally, atmospheric pressure (AP) IR-MALDI, which is applicable to liquid-state samples, is a promising technique to directly analyze untreated samples. Herein we perform AP-IR-MALDI mass spectrometry of a peptide, angiotensin II, using a mid-IR tunable laser with a tunable wavelength range of 5.50–10.00 µm and several different matrices. The wavelength dependences of the ion signal intensity of [M + H]+ of the peptide are measured using a conventional solid matrix, α-cyano-4-hydroxycinnamic acid (CHCA) and a liquid matrix composed of CHCA and 3-aminoquinoline. Other than the O–H stretching and bending vibration modes, the characteristic absorption peaks are useful for AP-IR-MALDI. Peptide ions are also observed from an aqueous solution of the peptide without an additional matrix, and the highest peak intensity of [M + H]+ is at 6.00 µm, which is somewhat shorter than the absorption peak wavelength of liquid water corresponding to the O–H bending vibration mode. Moreover, long-lasting and stable ion signals are obtained from the aqueous solution. AP-IR-MALDI using a 6–7 µm-band IR tunable laser and solvents as the matrix may provide a novel on-line interface between liquid chromatography and mass spectrometry. Copyright © 2015 John Wiley & Sons, Ltd.

Background Pseudomonas aeruginosa (PA) frequently develops antibiotic-resistant characteristics, which is clinically problematic. The main reason behind the rise of antibiotic-resistant PA is the extensive use of antibiotics. Therefore, a novel technique is needed to treat PA infections. Photodynamic therapy (PDT) is thought to have the potential to be a non-antibiotic treatment for infections. 5-Aminolevulinic acid (ALA), which works as a photosensitizer after being metabolized into protoporphyrin IX (PpIX) in the heme synthetic pathway, is used for PDT. Thus far, the in vivo effectiveness of PDT using ALA against PA is unknown. Objective In this study, we investigated PDT using ALA both in vitro and in vivo. Methods and results Although PDT with ALA alone did not show a bactericidal effect on PA, PDT with both ALA and EDTA-2Na had a bactericidal effect in vitro. In in vivo experiments, wounds healed faster in PA-infected mice treated with PDT using both EDTA-2Na and ALA compared to non-PDT. Conclusion These results suggest that PDT with EDTA-2Na and ALA is a potential novel treatment option for PA-infected wounds.

The interstitial photodynamic therapy (iPDT) with 5-aminolevulinic acid (5-ALA) is a safe and feasible treatment modality of malignant glioblastoma. In order to cover the tumour volume, the exact position of the light diffusers within the lesion is needed to decide precisely. The aim of this study is the development of evaluation method of treatment volume with 3D Monte Carlo simulation for iPDT using 5-ALA. Monte Carlo simulations of fluence rate were performed using the optical properties of the brain tissue infiltrated by tumor cells and normal tissue. 3-D Monte Carlo simulation was used to calculate the position of the light diffusers within the lesion and light transport. The fluence rate near the diffuser was maximum and decreased exponentially with distance. The simulation can calculate the amount of singlet oxygen generated by PDT. In order to increase the accuracy of simulation results, the parameter for simulation includes the quantum yield of singlet oxygen generation, the accumulated concentration of photosensitizer within tissue, fluence rate, molar extinction coefficient at the wavelength of excitation light. The simulation is useful for evaluation of treatment region of iPDT with 5-ALA.

Recently, combined therapy using chemotherapy and photodynamic therapy (PDT) has been proposed as a means of improving treatment outcomes. In order to evaluate the efficacy of combined therapy, it is necessary to determine the distribution of the anticancer drug and the photosensitizer. We investigated the use of imaging mass spectrometry (IMS) to simultaneously observe the distributions of an anticancer drug and photosensitizer administered to cancer cells. In particular, we sought to increase the sensitivity of detection of the anticancer drug docetaxel and the photosensitizer protoporphyrin IX (PpIX) by optimizing the ionization-assisting reagents. When we used a matrix consisting of equal weights of a zeolite (NaY5.6) and a conventional organic matrix (6-aza-2-thiothymine) in matrix-assisted laser desorption/ionization, the signal intensity of the sodium-adducted ion of docetaxel (administered at 100 μM) increased about 13-fold. Moreover, we detected docetaxel with the zeolite matrix using the droplet method, and detected PpIX by fluorescence and IMS with α-cyano-4-hydroxycinnamic acid (CHCA) using the spray method.

Although various theoretical models of matrix-assisted laser desorption/ionization (MALDI) have been proposed, no model can explain all of the experimentally observed phenomena. It has been reported that water content in the solvent used in the sample preparation has influence on the ion yield of MALDI. However, the previously proposed models do not take account of the solvent. Therefore, we have investigated the influence of the solvent on MALDI mass spectrometry of peptides. Among the solvents of water, methanol, ethanol, tetrahydrofuran, and acetonitrile, the signal peak area of the peptide ion was the largest when the water solvent was used. It is suggested that the residual water solvent contained in the dried matrix crystal has influence on the ionization processes of MALDI. It is inferred that inclusion of the influence of the residual solvent into the theoretical model of MALDI could contribute to the improvement of the precision of the model.

Background and Aims: There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Materials and Methods: Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormone-antagonistic human prostate cancer cells (PC-3) were evaluated. Results and Conclusions: Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated.

To investigate the correlation between the wavelength dependence of ionization threshold fluence of target molecule in matrix-assisted laser desorption/ionization by infrared (IR) laser and the IR absorption spectrum of matrix molecule, we have analyzed the IR absorption spectra of four matrix molecules using density functional theory and correlated ab initio molecular orbital method. The calculated IR absorption spectra of the isolated molecules showed more qualitative correlation with the wavelength dependence of ionization threshold fluence than those of the solid state structures. We can consider that a portion of matrix molecules lost the ordered crystal structure and that the transition to the diluted or isolated state occurred at the early process of IR laser irradiation. © 2012 Wiley Periodicals, Inc.

The optical properties of human brain tumor tissues, including glioblastoma, meningioma, oligodendroglioma, and metastasis, that were classified into "strong," "vague," and "unobservable" fluorescence by a neurosurgeon were measured and compared. The optical properties of the tissues were measured with a double integrating sphere and the inverse Monte Carlo technique from 350 to 1000 nm. Using reasons of ex-vivo measurement, the optical properties at around 420 nm were potentially affected by the hemoglobin content in tissues. Significant differences were not observed between the optical properties of the glioblastoma regions with "strong" and "unobservable" fluorescence. Sections of human brain tumor tissue with "strong" and "unobservable" fluorescence were stained with hematoxylin and eosin. The cell densities [mean ± standard deviation (S.D.)] in regions with "strong" and "unobservable" fluorescence were 31 ± 9 × 102 per mm2 and 12 ± 4 × 102 per mm2, respectively, which is a statistically significant difference. The higher fluorescence intensity is associated with higher cell density. The difference in cell density modified the scattering coefficient yet it does not lead to significant differences in the reduced scattering coefficient and thus does not affect the propagation of the diffuse fluorescent light. Hence, the false negatives, which mean a brain tumor only shows "unobservable" fluorescence and is hence classified incorrectly as nontumor, in using 5-ALA for detection of human glioblastoma do not result from the differences in optical properties of human brain glioblastoma tissues. Our results suggest that the primary cause of false negatives may be a lack of PpIX or a low accumulation of PpIX.

A new tandem time-of-flight mass spectrometer with an electrospray ionization ion source 'ESI-TOF/quadTOF' was designed and constructed to achieve the desired aim of structural elucidation via high-energy collision-induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration-type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic-field ion mirror and a microchannel plate detector. High-energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a-, d-, v- and w-type ions, characteristic of high-energy CID, were clearly observed in these spectra. These high-energy CID fragment ions enabled confirmation of the complete sequence, including leucine-isoleucine determinations. It was demonstrated that high-energy CID of multiply protonated peptides could be achieved in the ESI-TOF/quadTOF.

Relative intensities of singly-charged mono-bromide silver clusters Ag xBr+ formed from sputtering of a pressed pellet of silver bromide were measured by mass spectrometry. The obtained results suggest that the Ag xBr+ clusters have a structural formula of the form Ag x–1+(AgBr). The relative stability of Ag x–1+(AgBr) was determined by the intrinsic stability of the remaining metallic portion of the cluster (Ag x–1+) as predicted by the spherical jellium model (SJM). Unimolecular and high-energy collision-induced dissociation (CID) spectra of Ag xBr+ ( x = 2, 4, 6, 8, 10) clusters were also measured. In all of the spectra, the most intense fragment peaks were assigned to the Ag x–1+ ions accompanying the loss of AgBr. The difference in the relative intensities of the Ag x–1+ peaks between unimolecular dissociation and CID spectra led us to conclude that the weakest bond in the excited cluster Ag xBr+* is the Ag x–1+–AgBr bond and the structure of Ag xBr+ is a metallic Ag x–1+ ion cluster adduct with AgBr. The primary fragments observed in the CID spectra were also explained by the stabilities of the generated ion products and neutral fragments, both having even delocalized valence electrons. The present results were consistently explained by SJM. The dissociation behavior of Ag2Br+ can be explained on the basis of the calculated thermochemical data.

A JEOL JMS-HX110 double-focusing mass spectrometer was combined with the collision cell and spherical electric sector of a HITACHI M80-B instrument to perform tandem mass spectrometry experiments. A quadrupole lens doublet was mounted between the mass spectrometer and the collision cell to improve ion transmission into the collision cell. The detection system was also improved to expand the mass range of product ions. These improvements provided reliable spectra of high-energy electron transfer dissociation (HE-ETD) of peptides using an alkali metal target. The present results demonstrated that HE-ETD by a single collision with an alkali metal target could determine the amino acid sequence and phosphorylation site of peptides.

A novel design concept and the status of development of a matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometer in the stigmatic mode utilizing a perfect focusing property of a multi-turn time-of-flight (TOF) ion optics is reported. A multi-turn TOF ion optics consisting of four toroidal electric sectors has ideal features for a mass analyzer of stigmatic imaging mass spectrometry. The constructed multi-turn TOF mass analyzer, MULTUM-IMG, demonstrated a strong focusing property allowing MALDI-generated ions to revolve in a closed orbit for hundreds turns. Images of ion ensembles extracted from matrix surfaces patterned by several sizes of thin grid meshes (100–1000 lines/in.) or from arrays of small pigment dots (approximately 30 μm in diameter) fabricated on Si plates were examined by attaching an imaging detector to a MALDI ion source directly, and were found to retain the original spatially distributing patterns of the samples. Images of a patterned ion ensemble after multiple revolutions in MULTUM-IMG were examined, and were found to retain the images at the entrance of the multi-turn TOF section. The possibility of the novel stigmatic MALDI TOF imaging mass spectrometer has been proven by examining the building blocks.

Infrared-induced alignment change with wavelength tunable CO 2 laser irradiation for columnar liquid crystal domains was investigated for a liquid crystalline triphenylene derivative. A uniformly aligned alignment change of domains was observed when a chopped linearly polarized infrared laser light corresponding to the wavelength of the aromatic C–O–C stretching vibration band (9.65 μm) was irradiated. The results strongly imply that the infrared irradiation is a possible technique for device fabrication by use of columnar mesophase as a liquid crystalline semiconductor.

Matrix-assisted laser desorption and ionization by infrared laser (IR-MALDI) is expected to be an effective methods for soft-ionization of high-molecular weight proteins and intracellular proteins. IR-MALDI is not widely used because its low sensitivity, complexity, high cost, and as it does not work well on commercial MALDI time-of-flight mass spectrometers (TOFMSs). We employed a tunable mid-infrared (MIR) laser as a light source for MALDI to investigate the IR-MALDI. The laser wavelength can be tuned within a range from 5.5 to 10.0 μm, and included several biomaterial group vibration modes. We evaluated the wavelength dependence of ionization in IR-MALDI for four matrices: succinic acid, urea, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) and 2,5-dihydroxybenzoic acid (DHB). These matrices contained various groups of vibration modes, and absorbed an infrared (IR) energy at a specific wavelength. The mass spectra of angiotensin II was obtained at a specific wavelength corresponding to the C O stretching and benzene ring vibration mode. In IR-MALDI, we considered the strong molecular bond attracting an electron from a neighboring hydrogen atom, possibly protonating the hydrogen atom.

In this study, the alignment change with circularly polarized infrared irradiation for columnar mesophase was investigated for a liquid crystalline triphenylene derivative in order to get uniform domain with homeotropic alignment of columnar liquid crystal. It was found that circularly polarized infrared irradiation provides alignment change to form new domain with uniformly homeotropic (“face-on”) alignment and this phenomena is by way of the excitation of the selected vibrational mode of a chemical bond. The results strongly imply that the infrared irradiation is a possible technique for device fabrication by use of columnar liquid crystalline semiconductors.

Various dissociation channels of silver bromide cluster ion Ag2Br+ and silver cluster ion Ag3 + were observed in high-energy collisionally-activated dissociation (CAD) using a Cs target. The fragment patterns of the high-energy CAD were compared with those of the metastable dissociation and low-energy CAD. The difference in the fragment patterns between the high-energy CAD and the other dissociation methods was explained in terms of the internal energy distributions. The dissociation mechanisms of neutral silver bromide cluster Ag2Br and silver cluster Ag3 were also investigated by charge inversion mass spectrometry using the Cs target. While the fragment ions AgBr- and Ag2 - were dominantly observed in the charge inversion spectrum of Ag2Br+, the undissociated ion Ag3 - was observed as a predominant peak in the case of Ag3 +. The dissociation behavior of Ag2Br* can be explained on the basis of the calculated thermochemical data. Contrary to this, the predominant existence of the undissociated Ag3 - cannot be explained by the reported thermochemical data. The existence of undissociated Ag3 - suggests that the dissociation barrier is higher than the internal energy of Ag3 * (theoretical: 1.03 eV, experimental: 2.31 eV) estimated from the ionization potentials of Ag3 and Cs.

Traditional treatment options for prostate cancer are insufficient to cure advanced drug-resistant prostate cancer. Thus, as an alternative form of cancer therapy, photodynamic therapy (PDT) has become the main subject of intense investigation as a possible treatment modality. In this study, ultraviolet-inactivated viral vector, called hemagglutinating virus of Japan envelope (HVJ-E) was utilized to establish an effective delivery system for photosensitizer. Lipidated protoporphyrin IX (PpIX lipid) was inserted in HVJ-E by centrifugation to create a new drug delivering system that allows selective accumulation of photosensitizers in cancer cells. To study in vitro drug release mechanism of porphyrus envelope, the ultra-high voltage electron microscope tomography was applied. Next, to evaluate the photodynamic efficiency of porphyrus envelope for hormone antagonistic prostate cancer cells (PC-3), uptake of porphyrus envelope derived PpIX lipid and PpIX induced from exogenously administered precursor of 5-aminolevulinic acid hydrochloride (5-ALA) were compared by measuring fluorescence intensity of PpIX. Finally, to evaluate the efficacy of porphyrus envelope-PDT, laser light at a wavelength of 405 nm was irradiated to PC-3 cells. As a result, incorporation of porphyrus envelope-derived PpIX lipid occurred via membrane fusion, giving the highest fluorescence intensity when compared to 5-ALA-induced PpIX. Also, results from PDT experiment revealed the 28.6 × 10 3 -fold and 206-fold increase in therapeutic efficacy when compared to those of PDT using 5-ALA induced PpIX and PpIX lipid, respectively. Our findings suggest how porphyrus envelope can induce efficient accumulation of PpIX lipid, which can enhance the therapeutic efficacy of PDT against hormone antagonistic prostate cancer.

Atherosclerosis is a primary cause of critical ischemic diseases like heart infarction or stroke. A method that can provide detailed information about the stability of atherosclerotic plaques is required. We focused on spectroscopic techniques that could evaluate the chemical composition of lipid in plaques. A novel angioscope using multispectral imaging at wavelengths around 1200 nm for quantitative evaluation of atherosclerotic plaques was developed. The angioscope consists of a halogen lamp, an indium gallium arsenide (InGaAs) camera, 3 optical band pass filters transmitting wavelengths of 1150, 1200, and 1300 nm, an image fiber having 0.7 mm outer diameter, and an irradiation fiber which consists of 7 multimode fibers. Atherosclerotic plaque phantoms with 100, 60, 20 vol.% of lipid were prepared and measured by the multispectral angioscope. The acquired datasets were processed by spectral angle mapper (SAM) method. As a result, simulated plaque areas in atherosclerotic plaque phantoms that could not be detected by an angioscopic visible image could be clearly enhanced. In addition, quantitative evaluation of atherosclerotic plaque phantoms based on the lipid volume fractions was performed up to 20 vol.%. These results show the potential of a multispectral angioscope at wavelengths around 1200 nm for quantitative evaluation of the stability of atherosclerotic plaques.

Infrared-induced alignment change of liquid crystal domains was investigated for plastic columnar mesophase of a liquid crystalline triphenylene derivative. A homeotropic alignment change of domains was observed when a circularly polarized infrared light with the same wavelength was irradiated. The results strongly imply that the infrared irradiation is a possible technique for device fabrication by use of plastic columnar mesophase as a liquid crystalline semiconductor.

Infrared photoinduced change of discotic liquid crystal domains was investigated for a hexagonal columnar mesophase of a triphenylene derivative with three wavelengths of irradiation. The formation of uniform domains was observed under the irradiation with the wavelength of certain absorption bands of the compound. The technique could provide a novel technology to control the columnar alignment of viscous mesophase materials.

A novel matrix-assisted laser desorption/ionization (MALDI) method was developed for insoluble protein analysis. Solubilizing agents, such as 8 M urea, used for preparation of insoluble proteins do not absorb typical desorption lasers and impede transient laser heating in the conventional MALDI process. A mid-infrared free-electron laser (MIR-FEL) can individually activate various chemicals by tuning the MIR-FEL wavelength to selectively excite a vibrational mode. In the present method designated UV/FEL-MALDI, the same position on a sample deposit containing a denaturant at high concentration is exposed to a nitrogen laser pulse which is absorbed by UV-matrix and a MIR-FEL macropulse which is absorbed by denaturant in the same time frame. This scheme lets a denaturant at high concentration to be used for the MALDI sample preparation of insoluble proteins. The simultaneous irradiation of a nitrogen laser and MIR-FEL achieves spatially and temporally defined desorption, which is essential to TOFMS detection, while specificity and selectivity owing to the MIR-FEL wavelength can be conserved. The ability of UV/FEL-MALDI to detect the analyte which is deeply embedded in denaturant was examined using model protein samples dissolved into 8 M urea solution. In comparison to the conventional UV-MALDI, orders-of-magnitude improvement in signal-to-noise ratio was obtained by UV/FEL-MALDI with the MIR-FEL wavelength tuned around the absorption maximum of urea. The intact protein of human hair keratin, which is extremely insoluble and is not amenable to UV-MALDI and any other ionization methods, was subjected to UV/FEL-MALDI-TOFMS analysis. The molecule-related cluster ions of γ-keratin were detected for the first time.

Phosphorylation and dephosphorylation, which are the most remarkable post-translational modifications, are considered to be important chemical reactions that control the activation of proteins. First, we examine the phosphorylation analysis method by measuring the infrared absorption peak of the phosphate group that is observed at about 1070 cm−1 (9.4 urn) with Fourier Transform-Infrared Spectrometer (FT-IR). Next, we attempt to control the quantity of phosphorylation, that is to say an action like dephosphorylation without enzyme reactions, by irradiating 9.4 um-Free Electron Laser (9.4 um-FEL). FEL irradiation has an effect of some kind on the organization of infrared absorption of a phosphate group. We would now like to go on to develop this photochemical reaction like dephosphorylation by examining under several conditions and in detail. © 2004 Elsevier B.V. All rights reserved. PACS: 41.60.Cr;42.62.Be

Backgrounds/aims: To evaluate treatment with high peak power pulse energy by femtosecond ultrashort pulse laser (titanium sapphire laser) delivered at an 800 nm wavelength for corneal neovascularisation using photodynamic therapy (PDT) mediated by indocyanine green (ICG). Methods: Using a gelatin solid as an in vitro corneal model, the safety of laser power was studied to determine if it degenerated gelatin with or without ICG. The authors then induced corneal neovascularisation in rabbit eyes by an intracorneal suturing technique. Fluorescein angiography was used to evaluate occlusion before PDT and 0, 1, 3, and 10 days after PDT. The authors performed light microscopy with haematoxylin eosin staining and transmission electron microscopy to determine thrombosis formation in the neovascular regions. Results: The threshold of peak laser power density ranged from 39 to 53 W/cm 2 . Laser irradiation was started 30 seconds after a 10 mg/kg ICG injection, and all irradiated segments were occluded at 0, 1, 3, and 10 days at 3.8 J/cm 2 . Light and electron microscopy documented thrombosis formation in the neovascular region. Conclusion: Femtosecond pulse laser enhanced by ICG can be used for PDT. Because of effective closure of corneal neovascularisation at a low energy level, the high peak power pulse energy of the femtosecond pulse laser might be more efficacious than continuous wave laser for use with PDT.

Polarized infrared photoinduced change of liquid crystal domains was investigated for a hexagonal columnar phase of a liquid crystalline triphenylene derivative. A uniform and anisotropic change of domains was observed when a polarized infrared light corresponding to the wavelength of the absorption band of triphenylene core was irradiated. IR absorption dichroism is induced as a result of the reorientation of triphenylene core. The texture observation and polarized microscopic FT-IR spectra show that a change of the molecular alignment occurred and the direction of columns depends on the polarization direction of an actinic infrared light. The technique could provide a novel technology to control the columnar alignment of supra-molecular systems.

Phosphorylation and dephosphorylation that are most remarkable posttranslational modifications are important to the activation and deactivation of protein in living system. In the first approach, we showed first that it is possible to distinguish a phosphate group in a peptide using Fourier Transform Infrared Spectrometer (FT-IR). In this study, we used Free Electron Laser (FEL) at the wavelength of 9.4 μm corresponding to the peak of infrared absorption spectra derived from a phosphate group in phosphorylated peptides to explore the irradiation effect of FEL on the phosphorylation of a peptide (experimental condition). This study demonstrated that the FEL irradiation had effects of some kind on the organization of infrared absorption of a phosphate group. The promotion of the phosphorylation by FEL irradiation is supposed via the excitation of both protein and phosphate reactant.

The infrared photoinduced alignment change of liquid-crystal domains was investigated for a hexagonal columnar mesophase of a liquid-crystalline triphenylene derivative. A uniform and anisotropic alignment change of domains was observed when a polarized infrared (IR) light corresponding to the wavelength of the aromatic C–C stretching absorption band of the triphenylene core was used to irradiate the sample. The relationship between the aligned azimuthal angle of the columnar axis and the polarization of the IR incident irradiation was investigated. IR absorption dichroism is induced as a result of the reorientation of triphenylene core. Texture observation and polarizing microscope FTIR spectra show that a change of the molecular alignment occurred and that the direction of columns depends on the polarization angle of the IR light used for irradiation. The mechanism of the alignment change in a columnar liquid crystal film by IR irradiation is also discussed. The technique could provide a novel technology to control the columnar alignment of highly viscous liquid crystals.

Infrared photo-induced alignment change of liquid crystal domains was investigated for a hexagonal columnar mesophase of liquid crystalline triphenylene derivative. A uniform and anisotropic change of domains was observed when a polarized infrared light corresponding to the wavelength of the absorption band of triphenylene core was irradiated. IR absorption dichroism is induced as a result of the reorientation of triphenylene core part. The texture observation and polarized microscopic FT-IR spectra show that a change of the molecular alignment occurred, and the direction of columns depends on the polarizing angle of an irradiated infrared light. This technique could provide a novel technology to control the columnar alignment of supra-molecular systems.