Modified optical coefficient measurement system for bulk tissue using an optical fiber insertion with varying field of view and depth at the fiber tip.

To measure the few millimeter-scale macroscopic optical properties of biological tissue, including the scattering coefficient, while avoiding the instability that originates from sample slicing preparation processes, we performed propagated light intensity measurements through an optical fiber that punctures the bulk tissue while varying the fiber tip depth and the field of view (FOV) at the tip; the results were analyzed using the inverse Monte Carlo method. We realized FOV changes at the fiber tip in the bulk tissue using a variable aperture that was located outside the bulk tissue through a short high-numerical aperture (high-NA) multi-mode fiber with a quasi-straight shape. Using a homogeneous optical model solution, we verified the principle and operation of the constructed experimental system. A 200-µm-core-diameter silica fiber with NA of 0.5 and length of 1 m installed in a 21G needle was used. The detection fiber's shape was maintained over a radius of curvature of 30 cm. The dependences of the detected light intensity on the FOV and the depth showed better than 1.4% accuracy versus calculated dependences based on the measured optical properties of the solution. Adaptation of the method for use with complex structured biological tissue, particularly in the presence of a thick fascia, was not completely resolved. However, we believe that our specific fiber puncture-based measurement method for use in bulk tissue based on variation of the FOV with inverse Monte Carlo method-based analysis will be useful in obtaining optical coefficients while avoiding sample preparation-related instabilities.

Development of a practical animal model of photodynamic therapy using a high concentration of extracellular talaporfin sodium in interstitial fluid: influence of albumin animal species on myocardial cell photocytotoxicity in vitro.

Photodynamic reaction-induced photocytotoxicity using talaporfin sodium is inhibited by serum proteins binding to talaporfin sodium. The serum albumin binding site for talaporfin sodium differs among animal species. To identify a practical animal therapeutic model, we studied the ability of human, canine, bovine, and porcine albumin to influence talaporfin sodium-induced photocytotoxicity in rat myocardial cells in vitro. Human, canine, bovine, and porcine serum albumins were used. The ratio of talaporfin sodium binding, which is strongly associated with photocytotoxicity, was measured by ultrafiltration with an albumin concentration of 0.5-20 mg/ml and 20 µg/ml talaporfin sodium to mimic interstitial fluid. Rat myocardial cell lethality was measured by the WST assay 2 h after samples were exposed to a radiant exposure of 20 J/cm2 by a red diode laser (Optical Fuel™, Sony, Tokyo, Japan) with a wavelength of 663 nm. The binding ratio dependence on albumin concentration differed among the animal species. Bovine albumin exhibited the largest difference from human albumin, with a maximum difference of 31% at 2 mg/ml albumin. The cell lethality characteristic was similar between human and canine albumin. The cell lethality dependence on albumin was not in the same order as the binding ratio. Cell lethality was lowest for human albumin with higher albumin concentrations between 5 and 20 mg/ml. There were no significant differences in cell lethality between bovine and porcine albumin and between human and canine albumin. We suggest that the canine model may be a useful animal therapeutic model for evaluating photodynamic therapy using a high concentration of the photosensitizer in the extracellular space.

Effect of a photosensitization reaction performed during the first 3 min after exposure of rat myocardial cells to talaporfin sodium in vitro.

To better understand the mechanism of photodynamic cardiac ablation, we studied the effects of a photosensitization reaction (PR) performed during the first 3 min after rat myocardial cells were exposed to talaporfin sodium. A 3-mm-square microelectrode array with 64 electrodes was used to continuously measure the action potentials of the myocardial cells. A 30 µg/mL talaporfin sodium solution, a chlorine photosensitizer, was used, along with a 663-nm red diode laser (86 mW/cm2 for up to 600 s). The first trough of the measured action potential waveform corresponding to Na+ dynamics decreased exponentially with increasing PR duration. The average decay time of the exponential function from PR onset was 20.1 s. Marked morphological changes in the myocardial cells was observed after the PR. These results indicated that the behavior of the action potential waveform measured by the microelectrode array might be used as a less invasive method to evaluate the electrophysiological effects of a PR on myocardial cells.

Electrical superior vena cava isolation using photodynamic therapy in a canine model.

AIMS: With the new era of multi-tip radiofrequency or balloon ablation catheters replacing the point-to-point ablation strategy, we aimed to determine the feasibility of a ring-laser catheter ablation technology to electrically isolate the superior vena cava (SVC) by exploring the advantages of the limitless catheter tip size possibly with the photodynamic therapy (PDT)-mediated ablation. METHODS AND RESULTS: We developed a first-generation prototype of a circular-laser-mapping catheter by fitting a 7 cm plastic optical fibre onto a circular variable-loop Lasso™ mapping catheter. Following SVC venography, both the laser catheter and another ring catheter for monitoring the SVC potentials were placed at the SVC. After the systemic infusion of a photosensitizer (talaporfin sodium), we initiated the irradiation with an output of 1 W in three canines and 0.3 W in four. The creation of electrical isolation as well as occurrence of phrenic nerve injury, sinus node injury, and SVC stenosis were evaluated before, immediately after, and 1 month after the procedure. A PDT-mediated SVC isolation was successfully performed in all seven canines. The isolation was completed with a laser irradiation of 70.4 ± 71.4 J/cm under 30.9 ± 5.0 µg/mL of a photosensitizer without any sinus node injury, phrenic nerve palsy, or SVC stenosis in both the acute and chronic evaluations. The minimum isolation time of 270 s was not correlated with the laser input power or the photosensitizer concentration. CONCLUSION: The electrical SVC isolation was successfully and instantly achieved using the PDT laser-ring catheter without any complications.

Optimal conditions for cardiac catheter ablation using photodynamic therapy.

AIMS: Photodynamic therapy (PDT) is based on non-thermal injury mediated by singlet oxygen species and is used clinically in cancer therapy. In our continuing efforts to apply this technology to cardiac catheter ablation, we clarified the optimal condition for creating PDT-mediated lesions using a laser catheter. METHODS AND RESULTS: In a total of 35 canines, we applied a laser directly to the epicardium of the beating heart during open-chest surgery at 15 min after administration of a photosensitizer, talaporfin sodium. We evaluated the lesion size (depth and width) using hematoxylin-eosin staining under varying conditions as follows: laser output (5, 10, 20 W/cm(2)), irradiation time (0-60 s), photosensitizer concentration (0, 2.5, 5 mg/kg), blood oxygen concentration (103.5 ± 2.1 vs. 548.0 ± 18.4 torr), and contact force applied during irradiations (low: <20 g, high: >20 g). A laser irradiation at 20 W/cm(2) for 60 s under 5 mg/kg (29 µg/mL) of photosensitizer induced a lesion 8.7 ± 0.8 mm deep and 5.2 ± 0.2 mm wide. The lesion size was thus positively correlated to the laser power, irradiation time, and photosensitizer concentration, and was independent of the applied contact force and oxygen concentration. In addition, the concentration of the photosensitizer strongly correlated with the changes in the pulse oximetry data and fluorescence of the backscattering laser, suggesting that a clinically appropriate condition could be estimated in real time. CONCLUSION: Photodynamic therapy-mediated cardiac lesions might be controllable by regulating the photosensitizer concentration, laser output, and irradiation time.

Detailed in vitro study of the photosensitization reaction of extracellular Talaporfin sodium in rat myocardial cells.

BACKGROUND AND OBJECTIVE: We proposed a new non-thermal treatment for tachyarrhythmia that employs an extracellular photosensitization reaction. Oxygen depletion may easily occur in in vitro studies of this reaction because the photosensitizing agent is often highly concentrated in such studies. The aim of the current study was to examine the progress of the extracellular photosensitization reaction and the photocytotoxicity of extracellular Talaporfin sodium on myocardial cells for application in tachyarrhythmia therapy. MATERIALS AND METHODS: Photosensitization reactions were performed in single wells of 96-well plates; Talaporfin sodium solution concentrations from 5 to 40 µg/ml, radiant exposures up to 40 J/cm(2) , and irradiance of 0.29 W/cm(2) from a continuous wave (CW) red diode laser (wavelength: 663 nm) were used. We measured transient changes of temperature, photosensitizer fluorescence, dissolved oxygen pressure, and photosensitizer solution absorbance to monitor the progress of the photosensitization reaction in the system during laser irradiation. Rat myocardial cells were cultured in 96-well plates, and the drug-light interval was set to 15 minutes. We used a WST assay to measure cell lethality 2 hours after laser irradiation. RESULTS: A strong photosensitization reaction occurred several seconds after initiation of laser irradiation; this initial reaction depended upon dissolved oxygen. A gentler continuous photosensitization reaction followed the initial reaction, and was associated with temperature increases of less than 10°C. The oxygen pressure was kept in approximately 40 mmHg of the myocardial tissue oxygen pressure in the gentle photosensitization reaction phase. At radiant exposures from 10 to 40 J/cm(2) , a photosensitizer concentration of approximately 15 µg/ml was the threshold for myocardial cell necrosis in this in vitro system. The dependencies of photocytotoxicity on radiant exposure were separated into two distinct groups based on the molecular density ratio between Talaporfin sodium and albumin. CONCLUSIONS: This in vitro system for the extracellular photosensitization reaction may reflect the situation in live myocardial tissue. We found that the extracellular photosensitization reaction progressed in two distinctive phases; the first phase depended upon dissolved oxygen, and the second upon the molar density ratio between Talaporfin sodium and albumin. Cell lethality due to the extracellular photosensitization reaction was influenced by both of these factors in our in vitro system. We suggest that a photosensitizer concentration of 25 µg/ml might be necessary to treat myocardial tissue with therapies involving the extracellular photosensitization reaction.

Surface layer-preserving photodynamic therapy (SPPDT) in a subcutaneous mouse model of lung cancer.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) may be a less invasive treatment for lung cancer. Our newly developed surface layer-preserving PDT (SPPDT) technique enables us to irradiate deep tumor while preserving the overlying tissue. The aim of this basic study was to verify that the SPPDT technique might be applied to lung cancer. STUDY DESIGN/MATERIALS AND METHODS: PDT with talaporfin sodium was performed using a pulsed laser with different pulse dose rates (PDRs, 2.5-20.0 mJ/cm(2) /pulse) in a mouse model of subcutaneous tumor. To mimic the tracheal wall structure and a thoracic tumor in the tracheobronchus, we also made a mouse model in which a piece of swine cartilage was placed between the dermis and the tumor, and PDT was carried out 2 weeks after implantation. In both experiments, the tissue samples were collected 48 hours after PDT and evaluated microscopically. RESULTS: SPPDT using a high-PDR laser damaged the underlying tissue but left the superficial tissue intact in the mouse subcutaneous tumor model. In SPPDT, a higher PDR produced a thicker layer of intact superficial tissue than a lower PDR, while a lower PDR produced a deeper layer of damaged tissue than a higher PDR. SPPDT was also able to preserve the superficial tissue and to damage the tumor tissue beneath the cartilage implant. CONCLUSION: SPPDT was able to damage tumor beneath the superficial normal tissue layer, which included tracheal cartilage in the mouse model. The thickness control of SPPDT was provided by controlling laser pulse intensity. SPPDT is a new technology, whose future potential is unknown. The initial clinical application of this technology could be endoscopic treatment (e.g., palliative therapy of thoracic malignancies via bronchoscopy).

Study of blood charring precursor states using backscattering at 663 nm from blood and optical window boundary.

BACKGROUND AND OBJECTIVE: Contact laser irradiation is generally used in therapeutic laser procedures such as plastic surgery and laser catheter lead removal. However, it may induce blood charring on the surface of the optical window in blood circumstance so that the laser beam might be blocked. Various charring detection methods have been proposed, but they detect charring only after charring has occurred. This study investigates the transient behavior of red blood cells (RBCs) prior to the charring on the surface of an optical window during red laser irradiation in blood circumstance. MATERIALS AND METHODS: The backscattering light power was continuously measured to investigate the transient behavior of a 1-mm-thick porcine blood model (hematocrit: 40%) during continuous laser irradiation (center wavelength: 663 nm; irradiance: 81 W/cm(2)). A rabbit blood model was microscopically observed after irradiation. The absorption coefficient (µ(a)) and the reduced scattering coefficient (µ'(s)) were measured using a double integrating sphere setup and the inverse adding-doubling method. The backscattering light power was continuously measured in vivo during contact laser irradiation via a laser catheter in a porcine heart cavity. RESULTS: The results reveal that it may be possible to detect a precursory state of charring from a time course of the backscattering light power. µ(a) increased monotonically by 15% until charring occurred. µ'(s) decreased by 10% followed a broad peak until charring occurred. These changes in the optical property correspond to changes in the morphology of RBCs. Changes in the backscattering light power measured in vivo were similar to those measured ex vivo. CONCLUSIONS: The transient optical changes in blood prior to charring may be caused by changes in the morphology of RBCs on the optical window surface. Backscattering light power measurements may be a practical method to detect the precursor state of charring.

Myocardial electrical conduction block induced by photosensitization reaction in exposed porcine hearts in vivo.

BACKGROUND AND OBJECTIVE: This study proposes photosensitization reaction for non-thermal cardiac ablation in arrhythmia therapy. Acute and chronic phase experiments were conducted in exposed porcine hearts to demonstrate the photosensitization reaction-induced myocardial electrical conduction block in vivo. STUDY DESIGN/MATERIALS AND METHODS: The porcine left atrial appendage was exposed under an open-chest procedure. Then, a water-soluble chlorin photosensitizer, NPe6, was injected into the pigs intravenously at 5 or 10 mg/kg. About 15 or 30 minutes after the injection, a 663-nm continuous-wave diode laser was irradiated on the surface of the atrial appendage through a silica optical fiber. The laser energy was delivered to the tissue point by point at an energy density of 50-208 J/cm(2). RESULTS: Acute and chronic tissue damages as a result of the photosensitization reaction were determined by electrophysiology and histology, respectively. The change in the myocardial conduction time between two electrodes was measured immediately after the completion of the 35-mm irradiation line between the electrodes. The conduction delay of 35.5 milliseconds might be due to the change in the conduction pathway induced by transmural acute conduction block with the photosensitization reaction. The tissue temperature increase in the irradiated area was approximately 12.8°C. Azan-staining revealed about 1-mm transmural fibrosis of the atrial appendage at 2 weeks after the irradiation (50 J/cm(2)). CONCLUSIONS: The results suggest that the photosensitization reaction might induce acute and chronic myocardial electrical conduction block. Cardiac ablation with the photosensitization reaction might be a non-temperature-mediated methodology for arrhythmia therapy.

A three-compartment non-linear model of myocardial cell conduction block during photosensitization.

This study constructed a new non-linear model of myocardial electrical conduction block during photosensitization reaction to identify the vulnerable cell population and generate an index for recurrent risk following catheter ablation for tachyarrhythmia. A three-compartment model of conductive, vulnerable, and blocked cells was proposed. To determine the non-linearity of the rate parameter for the change from vulnerable cells to conductive cells, we compared a previously reported non-linear model and our newly proposed model with non-linear rate parameters in the modeling of myocardial cell electrical conduction block during photosensitization reaction. The rate parameters were optimized via a bi-nested structure using measured synchronicity data during the photosensitization reaction of myocardial cell wires. The newly proposed model had a better fit to the measured data than the conventional model. The sum of the error until the time where the measured value was higher than 0.6, was 0.22 in the conventional model and 0.07 in our new model. The non-linear rate parameter from the vulnerable cell to the conductive cell compartment may be the preferred structure of the electrical conduction block model induced by photosensitization reaction. This simulation model provides an index to evaluate recurrent risk after tachyarrhythmia catheter ablation by photosensitization reaction. A three-compartment non-linear model of myocardial cell conduction block during photosensitization.

Respiration and Heat Shock Protein After Short-Term Heating/Stretch-Fixing on Smooth Muscle Cells.

PURPOSE: A treatment device without a stent is needed for peripheral stenotic artery treatment. We have proposed short-term heating balloon angioplasty, photo-thermo dynamic balloon angioplasty (PTDBA). Though smooth muscle cells (SMCs) after PTDBA are fixed in a stretched formation in a porcine model, influences of this stimulus on SMCs have not been investigated. SMC migration after vascular dilatation would be related to chronic restenosis. The aim of this study was to examine respiratory activity and recovery ability of SMCs after short-term heating/stretch-fixing in vitro for chronic phase treatment effect discussion. METHODS: SMCs on a stretch chamber were heated for 15 s with stretching and fixed in a stretched formation. SMC migration is correlated with the cell respiratory activity. The amount of ATP production was measured using a WST-8 assay for respiratory activity evaluation. The intracellular expression of heat shock protein 70 was measured by an ELISA for recovery ability evaluation. RESULTS: In the case of 60 °C heating, SMC respiratory activity after short-term heating/stretch-fixing decreased drastically in all stretching rates. In the case of 50 °C heating, SMC respiratory activity decreased and then increased. Alternatively, the recovery ability at 60 °C was greater than that at 50 °C. CONCLUSIONS: SMCs heated at 60 °C with stretching would have high recovery ability and low respiratory activity related to SMC migration. These results may be important evidence in determining the treatment condition in PTDBA.

Clinical application of the mirror irradiation technique in photodynamic therapy for malignant glioma.

BACKGROUND: Intraoperative photodynamic therapy (PDT) using talaporfin sodium for malignant glioma is effective both in the experimental and in the clinical setting. Because the irradiation unit is fixed to the objective lens of the operating microscope, blind spots for irradiation exist. To overcome this problem, we developed a mirror reflecting system using a modified dental mirror. METHODS: The developed mirror is made of stainless steel, has a mirror-polished surface, and is rhodium coated on 1 side, which is the reflecting surface. The reflection rate was measured using He-Ne laser irradiation. The reflection intensity was measured using a laser power meter when the incident angle to the mirror was changed to 60°, 45°, and 30°, and the reflectance was calculated by the direct received light intensity from the laser. After confirming the safety of the fundamental experiment, PDT was performed with this developed mirror on 9 patients with malignant glioma (4 with recurrence and 5 newly diagnosed). RESULTS: The energy efficiency of the mirror was approximately 70 %, and apparent irregular reflection was not observed. Even during clinical use, apparent complications, such as irregular reflection, did not occur upon using the mirror in any of the patients. In all patients, recurrence did not occur in the site where mirror irradiation was performed, but in a deep site or a distant site to which sufficient laser irradiation did not reach. CONCLUSION: PDT using our newly developed mirror involves few instrumental changes compared with the conventional irradiation method, and is effective, safe, and inexpensive.

Diffused light attenuation at 664 nm for PDT in salted cadaver brain.

BACKGROUND: We investigated light attenuation at 664 nm, which is the excitation wavelength of photodynamic therapy (PDT) using talaporfin sodium, in a salted cadaver brain. Estimation of therapeutic lesions is important to ensure the effectiveness and safety of brain tumor PDT. Previously reported optical properties of the human brain vary widely. In this study, we measured the light attenuation in brain tissue using a practical method. We employed a salted cadaver brain, in which the mechanical and optical properties can be maintained as close as possible to those under operative conditions. METHODS: A neuroendoscope was inserted into the brain until the cerebral ventricle was reached. A thin cylindrical diffuser probe was advanced 10 mm from the endoscope tip. By another path from the brain surface, an optical fiber for measurement was inserted into a puncture needle, and a pair of needles was used to puncture the tissue and reach the same cerebral ventricle in which the endoscope tip was positioned. The attenuation of light intensities in the frontal lobe and cerebellum was measured by varying the bundle tip position. The starting positions of the bundle were confirmed by the endoscopic view. The measured light intensity attenuations were fitted with an exponential curve. RESULTS: The following attenuation coefficients were obtained: 0.20 ± 0.05 mm-1 in the cerebrum and 0.27 ± 0.05 mm-1 in the cerebellum. CONCLUSION: As conventional spectroscopic measurement may overestimate attenuation in the whole tissue, in situ measurement using the withdrawal technique might be appropriate for measurement of inhomogeneous biological tissues.

Continuous Optical Monitoring of Red Blood Cells During a Photosensitization Reaction.

Background: An oxygen-enriched photosensitizer solution was created by the addition of red blood cells (RBCs) as an investigative tool for photosensitization reactions (PRs). Although the oxygen levels and reaction progress can be monitored using the optical characteristics of hemoglobin, previously this has only been done using intermittent measurements. An increase in methemoglobin concentration with irradiation time was reported. Objective: We constructed a continuous optical measurement system to study the dynamics of the PR in a photosensitizer solution containing RBCs. We also measured the relationship between hemolysis and methemoglobin production in the solution. Materials and methods: A 664 nm wavelength continuous laser beam at 60 mW/cm2 was used to drive the PR, and a broadband (475-650 nm) light beam was used to monitor the absorption spectra during the PR. The light sources were arranged perpendicularly to cross at a 1 × 10 mm cuvette. The sample in this cuvette was prepared from a low-hematocrit rabbit RBC suspension medium containing 30 µg/mL talaporfin sodium, a chlorine photosensitizer. The concentrations of oxygenated hemoglobin, deoxygenated hemoglobin, and methemoglobin were obtained using a multiple regression analysis of the measured spectra. Results: The oxygen saturation decreased continuously during the PR. The relationship between the degree of hemolysis and produced methemoglobin concentration was confirmed. Conclusions: We determined the dynamics of the oxidation and oxygen desorption of hemoglobin, as well as RBC hemolysis, during the PR. Our measurement system, which uses the properties of hemoglobin contained in RBCs, might be useful for continuous monitoring of PR dynamics.

Oxygen-Enriched Photosensitizer Medium with Red Blood Cells to Study Tissue Interaction of Photosensitization Reaction.

BACKGROUND: It has been reported that the oxygen pressure of a photosensitizer medium decreases during an irradiation leading to decrease in the efficacy of the photosensitization reaction against the target cell in vitro. OBJECTIVE: The aim of this study was to obtain solutions with high dissolved oxygen levels in cultivated wells with perceiving oxygen environment and photosensitizer bleaching for photosensitization reaction studies. MATERIALS AND METHODS: We used a 10-mm-wide optical cell cuvette with a 1-mm optical path length as the well. A red blood cell (RBC) suspension with a hematocrit level of 0.625% was employed as the optical sample. The photosensitizer talaporfin sodium was added to a concentration of 30 µg/mL. The optical sample was irradiated by a 663-nm diode laser at 120 mW/cm2, for a total radiant exposure of 0-20 J/cm2, to induce a photosensitization reaction. Absorption spectra of the samples in the range of 475-700 nm were measured before and after each irradiation condition. Visible spectroscopy was selected to distinguish between the major three hemoglobin (Hb) types: oxygenated Hb, deoxygenated Hb, and met Hb. Also, this wavelength range was selected to investigate photobleaching using the Q band absorption peak. Each Hb concentration was estimated using a multiple regression analysis applied to the obtained absorption spectra. RESULTS: The relationship between oxygen saturation and the absorption peak in the Q band from the talaporfin sodium dynamics with increasing radiant exposure was revealed by our method with approximately twofold oxygen-dissolved solution. CONCLUSIONS: We could perceive the oxygen environment and the photosensitization reaction progression simultaneously with increasing dissolved oxygen by adding RBCs to the cell medium and measuring the absorption spectrum of it.

A Three-Compartment Pharmacokinetic Model to Predict the Interstitial Concentration of Talaporfin Sodium in the Myocardium for Photodynamic Therapy: A Method Combining Measured Fluorescence and Analysis of the Compartmental Origin of the Fluorescence.

To evaluate the effectiveness of photodynamic therapy occurring in the interstitial space of the myocardium, we estimated the interstitial concentration of talaporfin sodium in the canine myocardium by constructing a three-compartment pharmacokinetic model based on measured changes in talaporfin sodium plasma concentration and myocardial fluorescence. Differential rate equations of talaporfin sodium concentration in the plasma, interstitial space, and cell compartment were developed with individual compartment volume, concentration, and rate constants. Using measured volume ratios based on histological examinations, we defined that the myocardial fluorescence consisted of the linear addition of fluorescence generated from these three compartments. The rate constants were obtained by fitting to minimize the sum of the squared errors between the measured talaporfin sodium concentrations and the calculated concentrations divided by the number of data points using the conjugate gradient method in MATLAB. We confirmed that this fitting operation may be appropriate, because a coefficient of determination between the measured talaporfin sodium changes and the calculated concentrations using our equations was 0.99. Consequently, to estimate the interstitial concentration in the canine myocardium, we propose a three-compartment pharmacokinetic model construction methodology using measured changes in talaporfin sodium plasma concentration and changes in myocardial fluorescence.

Phototoxicity of Vascular Endothelial Cells Caused by Contact with Talaporfin Sodium for 15-120 Min: In Vitro and In Vivo Studies.

OBJECTIVE: To reveal the mechanism of vascular patency in the myocardium after photosensitization immediately after talaporfin sodium (TS) injection in a canine model, we investigated acute injury to vascular endothelial cells (VECs) in vitro and in vivo. BACKGROUND DATA: There are many reports of vascular shutdown within the target region in photodynamic therapy with TS. Vascular patency within healthy canine myocardium in which a photosensitization reaction starts immediately after injection of TS has been reported. MATERIALS AND METHODS: TS fluorescence in human umbilical vein endothelial cells and cell lethality were measured with drug contact time (DCT) up to 120 min at 20 µg/mL. Dependence of radiant exposure on cell lethality with 60 min DCT was investigated using two albumin concentrations that corresponded to those in plasma and interstices. Irradiation (21 mW/cm) outside the adventitia of canine cervical veins for 167 or 667 sec was emitted through a diffuser probe 30 min after intravenous injection of TS (2.5 mg/kg). Veins were extracted ∼30 min after the reaction and stained with von Willebrand factor. RESULTS: Intracellular fluorescence increased, but not cell lethality, with increasing DCT. Cell lethality increased gradually and reached 100% over 20 J/cm2 in the albumin concentration in the interstices. Normal VECs were found at the acute phase over 20 J/cm2 with a TS concentration in plasma of ∼14 µg/mL in vivo. CONCLUSIONS: VEC injury after a photosensitization reaction to healthy tissue shortly after TS injection might be low enough for the blood vessels to be patent.

Irradiance dependence of the conduction block of an in vitro cardiomyocyte wire.

BACKGROUND: To obtain therapeutic condition precisely by in vitro experiment, we studied the irradiance dependence of the electrical conduction blockage caused by a photodynamic reaction using a high extracellular concentration of talaporfin sodium on a novel in vitro cardiomyocyte electrical conduction wire. METHODS: The cardiomyocyte wires were constructed on patterned cultivation cover glass, which had cultivation areas 60µm in width, and a maximum length of 10mm. The talaporfin sodium concentration was set to 20µg/mL. The photodynamic reaction with a high extracellular photosensitizer concentration was performed with a short time interval (approximately 15min) between photosensitizer exposure and irradiation. A 663-nm laser was applied to the cardiomyocyte wire, and the irradiance was varied between 3 and 120mW/cm2. The cardiomyocyte electrical conduction was evaluated using the cross-correlation function of intracellular Ca2+ probe fluorescence brightness from an upper and lower section outside the laser irradiation area of a wire every 10s, which lasted up to 600s. RESULTS: The onset of electrical conduction blockage was defined by an 85% decrease in the cross-correlation function, compared with its initial value. The time for the electrical conduction blockage decreased from 600 to 300s as the irradiance was increased. Also, the probability of electrical conduction blockage was found to increase with increasing irradiance. CONCLUSIONS: We found a strong dependence on the irradiance for the time and probability of electrical conduction blockage.

Temperature Influence on Myocardial Cell Cytotoxicity of the Extracellular Photosensitization Reaction with Talaporfin Sodium and Serum Proteins at 17°-37°C.

BACKGROUND: We investigated the binding of talaporfin sodium with albumin and its photocytotoxicity during temperature changes by measuring absorbance spectra. The targeted tissue temperature differs according to the procedure. The photocytotoxicity efficiency should be investigated quantitatively because efficiency changes arising from temperature changes are expected. MATERIALS AND METHODS: The temperature dependence of talaporfin sodium binding with human serum albumin (0-20 mg/mL), high-density lipoprotein (0-0.04 mg/mL), and low-density lipoprotein (0-0.14 mg/mL) was investigated at 17°C, 27°C, and 37°C by measurement of absorbance spectra. Cell lethality was measured using a water-soluble tetrazolium-8 assay at 2 h after the photosensitization reaction at 17°C and 37°C. RESULTS: The binding ratios of talaporfin sodium with high-density lipoprotein decreased by 6.3% and those with low-density lipoprotein decreased by 12.8% when the temperature increased from 17°C to 37°C. Cell lethality increased significantly with a temperature rise from 17°C to 37°C at irradiation exposure of 20 and 40 J/cm2 and talaporfin sodium concentration of 20 µg/mL. CONCLUSIONS: From our in vitro data, we can predict that the change in photocytotoxicity efficiency would be negligible with a temperature decrease of <5°C from the body temperature in the case of photodynamic ablation with a short drug-light interval.

The Noninvasive Treatment for Sentinel Lymph Node Metastasis by Photodynamic Therapy Using Phospholipid Polymer as a Nanotransporter of Verteporfin.

Aim. The usefulness of photodynamic therapy (PDT) for treating sentinel lymph node (SLN) metastasis was evaluated. Materials and Methods. Verteporfin, a hydrophobic photosensitizer, forms a soluble aggregate with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). The concentrations of verteporfin were determined by measuring the fluorescence emitted at 700 nm. Seven days after the inoculation of A431 cells at the forearm of BALB/c nude mice, PMB-verteporfin was injected at dorsum manus and 75 J of light energy was delivered for 1 minute. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure, light exposure alone, PMB-verteporfin injection alone, and no treatment groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs, were harvested and evaluated. Results. The concentration of verteporfin in SLN was significantly higher than other organs. The combination of PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis (13%) comparing with no treatment group (52%), light exposure alone group (57%), and PMB-verteporfin injection alone group (46%). Conclusions. These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer and represent a potential alternative procedure to SLNB.