Vibrational spectroscopy for molecular characterisation and diagnosis of benign, premalignant and malignant skin tumours.

Understanding the molecular, cellular and tissue changes that occur during skin carcinogenesis is central to cancer research in dermatology. The translational aspects of this field--the development of clinical applications in dermatology from the laboratory findings--aim at improving clinical diagnosis, monitoring and treatment of skin cancer. Vibrational spectroscopy, both infrared (IR) and Raman spectroscopy, would be helpful in achieving those goals, since it has been shown to have potential in characterising and discriminating tumour and dysplastic tissue from normal tissue. Clinically differential diagnosis of skin tumours is often difficult and a histopathologic analysis of skin biopsies remains the standard for diagnostic confirmation. We review and update the literature on the subject, demonstrating that the IR and Raman spectra of skin tissues provide valid and useful diagnostic information about a number of skin tumours. We also include a survey of introduced sampling methods for IR and Raman spectroscopy in dermatology, and additionally describe the differences between microscopic, macroscopic and fibreoptic diagnosis of skin cancer. Although in its early stages, we remain optimistic that vibrational spectroscopy has the potential to be fully accepted as a rapid screening tool with sufficient sensitivity and specificity for non-destructive in vitro, ex vivo and in vivo analyses by the dermatological community. Further progress toward molecular characterisation of skin cancer by vibrational spectroscopy would have important research and clinical benefits in dermatology.

Fiber optic near-infrared Raman spectroscopy for clinical noninvasive determination of water content in diseased skin and assessment of cutaneous edema.

Currently, measuring Raman spectra of tissues of living patients online and in real time, collecting the spectra in a very short measurement time, and allowing diagnosis immediately after the spectrum is recorded from any body region, are specific advantages that fiber optic near-infrared Raman spectroscopy (NIR RS) might represent for in vivo clinical applications in dermatology. We discuss various methodological aspects and state of the art of fiber optic NIR RS in clinical and experimental dermatology to outline its present advantages and disadvantages for measuring skin in vivo, particularly its water content. Fiber optic NIR Fourier transform (FT) RS has been introduced to dermatological diagnostics to obtain information regarding the molecular composition of the skin up to several hundred micrometers below the skin surface in a relatively fast nondestructive manner. This has been especially important for probing for in vivo assessment of cutaneous (intradermal) edema in patients patch test reactions. Fiber optic NIR FT Raman spectrometers still require further technological developments and optimization, extremely accurate water concentration determination and its intensity calculation in skin tissue, and for clinical applications, a reduction of measurement time and their size. Another promising option could be the possibility of applying mobile and compact fiber optic charge-coupled device (CCD)-based equipment in clinical dermatology.

Photodynamic therapy of intestinal tumors with mono-L-aspartyl chlorin e6 (NPe6): a basic study.

BACKGROUND: To clarify the usefulness of the second-generation photosensitizer, mono-L-aspartyl chlorin e6 (NPe6), we examined the possibility of photodynamic diagnosis and photodynamic therapy for intestinal tumors in a mouse model. MATERIALS AND METHODS: NPe6 was intravenously administered to the tumor-bearing mice through the tail vein. The intestinal tumor sites were irradiated with a 664-nm diode laser at constant intervals after the administration of photosensitizers. The tumors were excised and fluorescence was observed in frozen sections by microscope. RESULTS: We observed the fluorescent image and calculated that the mean fluorescence intensity was significantly higher in the tumors than the normal mucosa during 6 hours (p < 0.05). The fluorescence of NPe6 was chiefly accumulated in the intestinal tumors as red fluorescence on the fluorescent microphotographic image. CONCLUSION: We conclude that NPe6 may be a valuable photosensitizer for the photodynamic diagnosis and photodynamic therapy of intestinal tumors.

Spectrometric characteristics and tumor-affinity of a novel photosensitizer: mono-l-aspartyl aurochlorin e6 (Au-NPe6).

Photodiagnosis and photodynamic therapy with photosensitizers can be indicated only for tumors of the superficial type, because these approaches utilizing visible light are limited by said light penetrability. To overcome this disadvantage, we innovated a novel photosensitizer, mono-l-aspartyl aurochlorin e6 (Au-NPe6), by incorporating a gold atom in the center of tetrapyrrole ring of NPe6 with a coordination bond. The gold atom in Au-NPe6 plays a role as an X-ray interceptor to detect deeply sited tumors. In this study, the absorption spectrum of novel Au-NPe6 in the diagnosis of deeply sited tumors was investigated, and the results were compared with the parent photosensitizer NPe6. Furthermore, the tumor-affinity of Au-NPe6 was evaluated using atomic absorption spectrometry. Despite the fact that both photosensitizers display a difference in the absorption spectrum, waveform changes of either photosensitizer with human serum albumin established a saturation point at a molar ratio of 1:1. The results indicate that it is highly possible that Au-NPe6 bound with albumin at a molar ratio (1:1) similar to NPe6. The accumulation rate of gold in tumor tissues was always significantly (p<0.05) higher than that in normal muscle tissues during the observation terms. Moreover, absorption spectra of tumor-tissue homogenates obtained from tumor-bearing mice after Au-NPe6 administration revealed a common peak with a wavelength equivalent to that of albumin-bond Au-NPe. This result suggests that the gold atom and NPe6 probably remained bonded even when Au-NPe6 was incorporated in tumor tissues.

Phase-resolved fluorescence study of mono-L-aspartyl chlorin E6.

The properties of a photosensitizer for photodynamic therapy, mono-L-aspartyl chlorin e6 (NPe6), were investigated using phase-resolved fluorescence. NPe6 was analyzed in water solution at concentrations ranging from 3.13x10(-7) to 8.00x10(-5) M. The photophysical parameters of the lowest singlet excited state of NPe6 molecules were experimentally determined. It was confirmed that NPe6 molecules were in the isolated molecular state at concentrations below 1.00x10(-5) M. It was also confirmed that the fluorescence in this concentration range was ascribable to the electronic transition of isolated NPe6 molecules from the lowest singlet excited state to the ground state. At concentrations above 1.00x10(-5) M, some of the NPe6 molecules formed dimers in water solution, which caused a red shift of the fluorescence spectrum and an enhancement of fluorescence in the 700-750 nm wavelength region. Semiempirical molecular orbital calculation revealed that the sodium aspartate attached to the tetrapyrrole ring through the ethanoic group was remarkably bent with respect to the tetrapyrrole plane. This bending appeared to hinder the formation of NPe6 dimers at concentrations up to 1.00x10(-5) M.

Photodynamic therapy with talaporfin sodium induces dose-dependent apoptotic cell death in human glioma cell lines.

OBJECTIVE: To investigate the kinetics of cell death in human glioma cell lines induced by photodynamic therapy (PDT) with the second-generation photosensitizer talaporfin sodium (TS) and a 664-nm diode laser. MATERIALS AND METHODS: Three human glioma cell lines (T98G, A172, U251) were studied. After incubation of the cell lines with various concentrations of TS for 4 h, PDT using diode laser irradiation at 33 mW/cm² and 10 J/cm² was performed. Cell viability and changes in cell morphology were examined by the Cell Counting Kit-8 assay and phase-contrast microscopy, respectively. In addition, to evaluate the pathology of cell death, changes in cell viability after treatment with a caspase activation inhibitor and an autophagy inhibitor were also examined. RESULTS: In all 3 human glioma cell lines, TS induced dose-dependent cell death. However, the 50% lethal dose of TS varied among these cell lines. The main morphological feature of cell death was shrinkage of the cell body, and the number of cells with this morphological change increased in a time-dependent manner, resulting in cell death. In addition, a dose-dependent improvement in cell viability by the caspase inhibitor Z-VAD-fmk was observed. CONCLUSION: PDT with TS induces dose-dependent apoptosis in human glioma cell lines. However, the sensitivity to PDT varied among the cell lines, indicating a possible difference in the intracellular content of TS, or a difference in the susceptibility to the intracellular oxidative stress caused by PDT.

Preliminary clinical report on safety and efficacy of photodynamic therapy using talaporfin sodium for malignant gliomas.

OBJECTIVES: To investigate the safety and efficacy of photodynamic therapy (PDT) using talaporfin sodium in patients with surgically, completely unresectable malignant gliomas with invasion into the eloquent areas of the brain associated with language and motor functions. MATERIALS AND METHODS: Subjects consisted of consecutive 14 adult patients with malignant gliomas that were shown on preoperative diagnostic imaging to have invaded the eloquent areas of the brain. Of these, 6 patients had newly diagnosed tumors and 8 patients had recurrent tumors. In 15 craniotomy and tumor resection procedures, PDT was used as additional intraoperative treatment 24 h after 40 mg/m(2) of talaporfin sodium had been injected intravenously. After the tumor bulk had been resected as extensively as possible either 1 or 2 sites of probable tumor invasion in the bottom of resection cavity were irradiated superficially with a 664-nm diode laser for 180 s (27 J/cm(2)) at a power density of 150 mW/cm(2). RESULTS: PDT achieved a response rate of 80% at the treated sites in the 6 patients with newly diagnosed malignant gliomas. In these patients, the median progression-free survival time was 23 months. The median survival time in 3 patients who died was 26 months, and the remaining 3 patients survived for more than 3 years with a good Karnofsky Performance Scale (KPS). In the 8 patients with recurrent tumors who received PDT, their response rate was low (25.0%), their gliomas recurred 3 months after PDT, and their survival time was only 9 months following PDT. No adverse events directly attributable to PDT occurred in any patients. Protection against light was only required for approximately 3 days after PDT. CONCLUSION: We examined the safety and efficacy of PDT using talaporfin sodium as an additional intraoperative treatment for malignant glioma. PDT in addition to surgical resection achieved better therapeutic results than conventional protocols, especially in patients with newly diagnosed malignant gliomas. However, the current methodology has some limitations with respect to patients with recurrent tumors. Larger-scale studies are required to confirm the clinical feasibility of PDT plus surgery.

X-ray fluorescence camera for imaging of iodine media in vivo.

X-ray fluorescence (XRF) analysis is useful for measuring density distributions of contrast media in vivo. An XRF camera was developed for carrying out mapping for iodine-based contrast media used in medical angiography. Objects are exposed by an X-ray beam from a cerium target. Cerium K-series X-rays are absorbed effectively by iodine media in objects, and iodine fluorescence is produced from the objects. Next, iodine Kalpha fluorescence is selected out by use of a 58-microm-thick stannum filter and is detected by a cadmium telluride (CdTe) detector. The Kalpha rays are discriminated out by a multichannel analyzer, and the number of photons is counted by a counter card. The objects are moved and scanned by an x-y stage in conjunction with a two-stage controller, and X-ray images obtained by iodine mapping are shown on a personal computer monitor. The scan pitch of the x and y axes was 2.5 mm, and the photon counting time per mapping point was 2.0 s. We carried out iodine mapping of non-living animals (phantoms), and iodine Kalpha fluorescence was produced from weakly remaining iodine elements in a rabbit skin cancer.

Embossed radiography utilizing energy subtraction.

Currently, it is difficult to carry out refraction-contrast radiography by using a conventional X-ray generator. Thus, we developed an embossed radiography system utilizing dual-energy subtraction for decreasing the absorption contrast in unnecessary regions, and the contrast resolution of a target region was increased by use of image-shifting subtraction and a linear-contrast system in a flat panel detector (FPD). The X-ray generator had a 100-microm-focus tube. Energy subtraction was performed at tube voltages of 45 and 65 kV, a tube current of 0.50 mA, and an X-ray exposure time of 5.0 s. A 1.0-mm-thick aluminum filter was used for absorbing low-photon-energy bremsstrahlung X-rays. Embossed radiography was achieved with cohesion imaging by use of the FPD with pixel sizes of 48 x 48 microm, and the shifting dimension of an object in the horizontal direction ranged from 100 to 200 microm. At a shifting distance of 100 mum, the spatial resolutions in the horizontal and vertical directions measured with a lead test chart were both 83 microm. In embossed radiography of non-living animals, we obtained high-contrast embossed images of fine bones, gadolinium oxide particles in the kidney, and coronary arteries approximately 100 microm in diameter.

Uptake and retention of the photosensitizer mono-L-asparthyl chlorine e6 in experimental malignant glioma.

The objective of the study was to investigate the potential of mono-L-aspartyl chlorine e6 (NPe6), a water-soluble photosensitizer derived from chlorophyll, for use in photodynamic diagnosis (PDD) of malignant brain tumor. A C6 glioma cell line was transplanted in the SD rat brain to create a brain tumor model. Five days after transplantation, NPe6 was administrated via the tail vein at concentrations ranging from 1.25 to 10 mg/kg; then the skull was opened in the rat brain, the site of tumor transplant was irradiated with a diode laser beam at 664 nm, and the time-course intensity and distribution of emerging fluorescence were observed. Furthermore, the correlation between fluorescence distribution and histopathological findings was investigated in the removed brain. Fluorescence was observed in the site of brain tumor transplant from 5 min after injection, and stable fluorescence was recognized at the site until 4 h after administration. No differences were noted in fluorescence intensity at NPe6 doses of 2.5 mg/kg or more; therefore, it was possible to estimate the optimal dose range. Fluorescence distribution had a clear correlation with tumor cell density, and it was possible to capture the margin of tumor cell invasion with fluorescence. The photosensitizer NPe6 is capable of assessing tumor cell density in malignant glioma tissue in terms of differences in fluorescence intensity. The usefulness of PDD using 5-aminoleveulinic acid during surgery for malignant glioma has been recognized in recent years. The results of the present study suggested the potential of NPe6 as a promising photosensitizer for use in PDD for accurate grasp of the extent of removal during the course of malignant glioma surgery.

Identification and characterization of skin biomolecules for drug targeting and monitoring by vibrational spectroscopy.

The article discusses the application of vibrational spectroscopy techniques for in vivo identification and characterization of glucose biomolecules monitored in the skin of healthy, prediabetes and diabetes subjects; for molecular characterization of water and proteins in in vivo monitored patch tested inflamed skin of the patients with contact dermatitis; for description of nucleic acids and proteins at the molecular level with progression to malignancy in skin cancerous lesions. The results of the studies show new possibilities to assess activity levels of glucose metabolism in the skin tissue of healthy, prediabetes and diabetes subjects; activity and severity of inflammation; activity of the processes of carcinogenesis with regard to benign, premalignant and malignant transformation. Based on our findings, we suggest that vibrational spectroscopy might be a rapid screening tool with sufficient sensitivity and specificity to identify and characterize skin biomolecules in described diseases for drug targeting and monitoring by the pharmacological community.

Photodynamic therapy of C6-implanted glioma cells in the rat brain employing second-generation photosensitizer talaporfin sodium.

OBJECT: The usefulness of photodynamic therapy (PDT) as a local therapy for malignant glioma was evaluated by investigating histological changes in a rat C6 glioma model treated with a combination of talaporfin sodium, a water-soluble photosensitizer derived from chlorophyll and exposure to a diode laser. METHODS: Glioma cells (C6) at the confluence stage were transplanted stereotactically into the right frontal lobe of SD rats. Five days later, the rats underwent right frontal craniotomy and intravenous administration of talaporfin sodium. One hour after talaporfin sodium administration, each rat was irradiated by a 664 nm diode laser beam. The brain was removed 1, 3 or 6h after laser irradiation for histological examination of tumor-affected brain tissue and surrounding normal brain tissue. RESULTS: In addition to the tumor mass, tumor cells invading surrounding edematous brain tissue were seen in untreated rats, ranging from the brain surface to a depth of 2mm. One hour after PDT, coagulation necrosis as well as disappearance of indication of cell viability such as disappearance of tumor cell processes and foamy changes of cytoplasm were noted in the tumor tissue at a depth of 0.5mm, accompanied by reduction of cytoplasmic glial fibrillary acidic protein (GFAP) expression and appearance of granular M30 cytodeath positivity. Three hours later, the cytoplasm of the residual tumor cells showed disappearance of GFAP expression and increased expression of M30 cytodeath. Six hours later, the foamy cytoplasm of swollen tumor cells demonstrated strong positivity for M30 cytodeath. CONCLUSION: PDT using talaporfin sodium induced coagulation necrosis and apoptosis in rats with C6 glioma.

Development of blood vessel viewer using multi spectral imaging technology.

Using multi spectral imaging technology, which enable us to simultaneously obtain spatial and wavelength information, we have newly developed blood vessel viewer. Our device is composed of the light source, the lens or the endoscope, the optical device, CCD camera, and PC. As an evaluation of our device, we measured the skin diffuse reflectance spectrum of the human hand. The blood vessel observed by our device was corresponding to an anatomy finding. However, even if Principal Component Analysis was used, the reflection of horny was not able to be removed completely.

Inspection of skin hemodynamics with hyperspectral camera.

Using hyperspectral imaging techniques, which enable us to simultaneously obtain spatial and wavelength information, we have improved upon the newly developed hyperspectral camera to develop a method of observing changes in skin melanin levels and hemodynamics over time. As an evaluation of this method, we measured the skin diffuse reflectance spectrum of the human middle finger in an experiment of blood flow blockage in the brachial region. The changes in skin hemodynamics observed through this method match the behavior expected based on clinical knowledge, and also show an extremely high correlation with results obtained by the Erythema Index, which is used to make similar calculations from a limited number of wavelengths.

Tumor viability using real-time spectral images.

PURPOSE: We observed real-time spectral images using line-scan imaging spectrography in order to evaluate tumor viability. METHODS: Japanese albino rabbits, which underwent the implantation of VX2 tumor strain, were used as subcutaneous tumor models (n = 54). Photodynamic therapy (PDT) consisted of semiconductor laser irradiation of the tumorous lesion. The experimental groups consisted of a PDT group (n = 15) and a non-PDT group (control group, n = 15). The spectral images taken by a CCD camera underwent computer processing. Next, the spectrum of the tumorous lesion was observed and the peak spectrum value was measured. RESULTS: Two peaks (545 and 575 nm) corresponding to the absorbance spectrum of oxygenated hemoglobin (oxyHgb) were observed in the untreated area. In the treated area, however, they disappeared and a different peak (560 nm) corresponding to the absorbance spectrum of deoxygenated hemoglobin was observed. PDT induced ischemic tissues and the cells could be confirmed in real time in vivo in monochrome and color images reflecting the oxyHgb amount. A histopathological examination and phosphotungstic acid hematoxylin staining demonstrated diffuse fibrin accumulation in the microvessels, while proliferating cell nuclear antigen staining showed a reduced nuclear stainability. CONCLUSIONS: These results demonstrated that the real-time spectral images showed the actual histological conditions such as a blocked tumor blood flow and reduced tumor viability.

Micro-optical fiber probe for use in an intravascular Raman endoscope.

We believe that we have developed the narrowest optical-fiber Raman probe ever reported, 600 microm in total diameter, that can be inserted into coronary arteries. The selection of suitable optical fibers, filters, and a processing method is discussed. Custom-made filters attached to the front end of a probe eliminate the background Raman signals of the optical fiber itself. The experimental evaluation of various optical fibers is carried out for the selection of suitable fibers. Measurement of the Raman spectra of an atherosclerotic lesion of a rabbit artery in vitro demonstrates the excellent performance of the micro-Raman probe.

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries.

Flexible treatments for intimal hyperplasia after angioplasty are still needed. The aim of this study was to demonstrate the long-term effects of vascular photodynamic therapy with talaporfin sodium on intimal hyperplasia following interventional injury. Intimal hyperplasia was induced by balloon distension injury to the carotid artery in 31 rabbits. Talaporfin, 5.0 mg/kg, was delivered systemically immediately after balloon injury. The injury site was irradiated with a diode laser light of wavelength 664 nm using a fluence of 50 J/cm2 after 30 min. At day 3 and weeks 3, 6, 9, 15, and 25 after photodynamic therapy, the treated artery of each rabbit was excised and examined immunohistochemically. Thirty minutes after talaporfin administration, drug fluorescence was found only in the balloon-injured carotid artery wall. At 3 days, no smooth muscle cells were seen in the media of the photodynamic therapy-treated arterial segments. Intimal hyperplasia developed progressively in the balloon-injured and untreated segments; however, in the segments treated with photodynamic therapy, intimal hyperplasia was markedly suppressed until 25 weeks and the media was repopulated by smooth muscle cells without macrophages. Vascular photodynamic therapy with talaporfin may be used to inhibit restenosis after vascular intervention.

Pilot study of topical delivery of mono-L-aspartyl chlorin e6 (NPe6): implication of topical NPe6-photodynamic therapy.

Photodynamic therapy (PDT) is an evolving cancer treatment with promising results in treating malignant tumors by photoactivation of a photosensitizer with a specific wavelength. The second generation photosensitizer mono-L-aspartyl chlorin e6 (NPe6) was reported to have significant efficacy in killing cancer cells in vitro and in vivo. Though topical application might yield a higher local concentration and less systemic side effect, no data concerning topical absorption of NPe6 is available even though the drug has already been used in clinical trial for several years. To evaluate the possibility of topical delivery of NPe6 via an animal model, escalated concentrations of NPe6 were applied to BALB/c mouse skin for a different time periods after barrier disruption with tape stripping. Since NPe6 fluorescence intensity and drug concentration in tissue was well correlated, we evaluated drug penetration depth with frozen sections of treated and non-treated skin under a fluorescence microscope. An on-line fluorescence imaging system was used to monitor the NPe6 fluorescence kinetics in the skin. The fluorescence microscope confirmed successful topical delivery of NPe6 in mouse skin with or even without barrier disruption. Orange to red NPe6 fluorescence appeared at the epidermis, dermis, and even the muscular layer when using 10 mg/ml NPe6 application. The fluorescence intensity peaked at 1 h and revealed a dose-dependent response pattern. NPe6 treated versus non-treated skin showed a statistically significant difference by Student's t-test (P<0.05). The results described here suggest that topical delivery of NPe6 is possible. It showed fast and deep penetration into mouse skin. This implies that NPe6 might be useful as a topical photosensitizer for PDT in treating skin cancers.

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy.

The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm(-1). These results suggest that exposure to the ELF magnetic field has reversible effects on the N-H inplane bending and C-N stretching vibrations of peptide linkages, and changes the secondary structures of alpha-helix and beta-sheet in cell membrane proteins.