Photodamage to mitochondria, endoplasmic reticulum (ER) or lysosomes can lead to activation of the apoptotic program, as can exposure of cells to the non-peptidic Bcl-2/Bcl-x_L antagonist HA14-1. Many signaling pathways are evoked by photodynamic therapy (PDT), presumably from oxidative stress effects. To discover which of the latter effects might be unique to PDT, we compared some photodynamic effects with HA14-1 treatment, using murine leukemia L1210 cells in culture. Two photosensitizers were employed: the porphycene CPO and the chlorin NPe6. The former targets the endoplasmic reticulum (ER) and causes Bcl-2 photodamage, while NPe6 targets lysosomes, resulting in protease-induced cleavage and activation of Bid to form the pro-apoptotic product t-Bid. PDT at either target will lead to loss of the mitochondrial membrane potential ΔΨ_m, translocation of cytochrome c to the cytosol and an apoptotic response. Photodynamic effects of CPO or NPe6 led to activation of several 'stress proteins' and intracellular oxidation of the probe dihydrodichlorofluorescein (H₂DCF). All of these effects were mimicked by HA14-1, indicating that these early responses to PDT result from initiation of apoptosis, however achieved. After CPO-catalyzed PDT or HA14-1 treatment, we observed a prompt release of Ca²⁺ into the cytosol, but this was insufficient to significantly alter mitochondrial calcium levels. The apoptotic response to HA14-1 or Bcl-2 photodamage was markedly promoted by the protein kinase C (PKC) inhibitor staurosporin (STS). These effects were not observed after photodamage catalyzed by NPe6, indicating that calcium release and PKC interactions are associated with loss of Bcl-2 function, but not Bid activation.

Angularly-resolved light scattering is an established method of particle sizing. Scattering from intact cells provides information about the size distributions of intracellular scatterers. Mitochondria are important light scatterers, especially at forward angles. Nuclei play an important part in scattering light at extreme forward angles and in backscattering geometries. Because changes in mitochondrial morphology are among the early responses to photodynamic therapy (PDT) using mitochondrial-localizing sensitizers and because these changes may be important in determining the fate of the cell, it is interesting to consider light scattering as a means of assessing the response of cells and tissue to PDT. Simple transmission measurements in an absorption spectrophotometer report a rapid reduction in scattering in cells subjected to aminolevulinic acid (ALA)-PDT. ALA-PDT with a fluence of 5 J cm^-2 induces a change in the angularly-resolved light scattering from EMT6 cells in suspension within approximately 45 minutes of irradiation. At earlier times following this fluence, the scattering differs only slightly from that observed with control cells. Analysis of the post-treatment scattering data at forward angles is consistent with mitochondrial swelling. Qualitatively similar changes in scattering are observed immediately after a fluence of 10 J cm^-2 in cells sensitized with Pc 4.

The lowest excited state of molecular oxygen, singlet molecular oxygen (a¹Δ_g), is generally regarded to be the active cytotoxic species in photodynamic therapy (PDT). As a result, the direct detection of singlet oxygen in biological systems should be of great value in elucidating the mechanisms underlying the observed effects of PDT. We have recently shown that singlet oxygen can be detected by its weak 1270 nm phosphorescence (a¹Δ_g→X³Σ_g^-) from a single nerve cell upon irradiation of a photosensitizer incorporated into the cell. In this paper, we discuss issues pertinent to the direct optical detection and imaging of singlet oxygen from single cells.

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1,000 neonates and 1 in 5,000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regiments of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (SA) grown onto kirschner wires (K-wire). SA-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of SA biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. SA infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg/Kg) intraperitoneally followed 4 hr later by light (635 ± 10 nm; 38 or 75 J/cm²) delivered transcutaneously via an optical fiber placed onto the tibia. In vitro, MB and ALA displayed similar cell kill with ≥ 4log₁₀ cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat SA cultures grown in vitro or in vivo using an animal model of osteomyelitis.

The syntheses and properties of novel water-soluble phthalocyanies are reported, as well as new chemistry of tetrabutanoporphyrins aimed at the preparation of new, functionalizable, tribenzoporphyrin systems. The objective of the study is to provide new second-generation photosensitizing drugs, with long-wavelength absorption, for use in the photodynamic therapy of tumors.

We are reporting elsewhere, the promising photodynamic effect of Hypericum perforatum L. extract (PMF) against T24, NBT-II tumor bladder cells, and HL-60 leukemic cells (using 630nm, and 530nm laser light respectively). The main advantages of the extract as a photosensitizer are its low cost, extensive availability, adequate solubility, minimal toxicity, and use with a range of wavelengths. Extraction of dry herb with methanol yields the methanolic extract (ME) in 11%, which is then fractionated using liquid / liquid extraction, yielding the polar methanolic fraction (PMF) in 9,9% overall yield. Hypericin, a photosensitizing ingredient of the herb, was found in these extracts in concentrations as low as 0,51%, and 0,57% respectively. Laser induced fluorescence spectra from the ME and PMF were recorded in order to evaluate their photodiagnostic capacity. An Argon-ion laser was employed for the excitation of the samples. It was shown that the extracts resulted in different fluorescence spectra related both to their intensity, and shape. The intensities of these spectra were only 8 times less compared to the fluorescence of pure hypericin. The dependence of the signal on the pH of the medium of pure hypericin and of PMF was also investigated in order to determine specific spectra variations. According to the results hypericin fluorescence signal fades smoothly in highly acidic medium, while it decreases sharply in highly basic environment. On the contrary PMF gives a slow decrease of fluorescence in both acidic and basic medium. These data suggest that PMF-induced fluorescence is highly sensitive in basic and acidic environment.

Many Type II photosensitizers are substrates of the singlet oxygen they generate. They are, therefore, converted to photoproducts when exposed to light in the presence of oxygen. While most photoproducts appear to be biologically inert, one of the photoproducts generated by selenomerocyanine photosensitizers is a form of elemental selenium that is surprisingly toxic to tumor cells if allowed to form conjugates with serum albumin or lipoproteins. Albumin and lipoproteins act as delivery vehicles for the cytotoxic entity, exploiting the fact that many tumor cells have an increased capacity to bind and internalize albumin and/or lipoproteins. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood but is obviously quite different from the singlet oxygen-mediated mechanism of merocyanine-mediated photodynamic therapy (PDT). Whereas merocyanine-PDT is directed against the plasma membrane, is more effective at 4 °C than at room temperature, and is inhibited by excess albumin and lipoproteins, selenomerocyanine-derived cytotoxic photoproducts act on intracellular targets, are ineffective at low temperatures, and require albumin or lipoproteins as carrier molecules. The discovery of cytotoxic Se(0)-protein conjugates provides a rare example of photoproducts contributing substantially to the cytotoxic effect of PDT and challenges the widely held notion that elemental selenium is biologically inert.

Some Gram-positive bacteria (including the causative agent of anthrax - Bacillus anthracis) survive conditions of stress and starvation by producing dormant stage spores. The spore’s multilayered capsule consists of inner and outer membranes, cortex, proteinaceous spore coat, and in some species an exosporium. These outer layers enclose dehydrated and condensed DNA, saturated with small, acid-soluble proteins. These protective structures make spores highly resistant to damage by heat, radiation, and commonly employed anti-bacterial agents. Previously Bacillus spores have been shown to be resistant to photodynamic inactivation (PDI) using dyes and light that easily destroy the corresponding vegetative bacteria, but recently we have discovered that they are susceptible to PDI. Photoinactivation, however, is only possible if phenothiazinium dyes are used. Dimethylmethylene blue, methylene blue, new methylene blue and toluidine blue O are all effective photosensitizers. Alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin and benzoporphyrin derivative are ineffective against spores even though they can easily kill vegetative cells. Spores of B. cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, while B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores and for which conventional sporicides would have unacceptable tissue toxicity.

A goal of our laboratory is to accurately define the parameters of light dose and drug dose that contribute to tissue destruction after Photodynamic therapy (PDT). Using Photofrin as sensitizer, we examined a range of drug doses, various intervals between injection and light treatment, and various fluence rates. The effect of Photofrin photosensitizer encapsulated in liposomal delivery vehicle was also studied. Three liposome delivery vehicles were chosen to deliver the photosensitizer in vivo: DPPC/cholesterol, DMPC/HPC and stealth liposomes. Tumor response and microvessel behaviour were examined in tumor and surrounding skin in a mouse model. Under these conditions, better selectivity of tissue damage was seen using some of the treatment. These data might be used to design better clinical protocols for patient care. In memory of Dr. Victor Fingar (Supported by R01 CA51771).

Monitoring singlet molecular oxygen (¹O₂) produced by photodynamic therapy (PDT) can lead more precise and effective cancer treatment. Physical Sciences Inc. (PSI) has developed a singlet oxygen monitor based on a pulsed diode laser technology. In this paper, we present results of singlet oxygen detection in the solution phase and in a rat prostate cancer cell line, as well as PDT mechanism modeling. We describe an improved detection approach for singlet oxygen monitoring that employs a fiber-coupled optical set-up and fast data acquisition system.

Refractory carcinoma in situ and resistant multifocal transitional cell carcinoma (TCC) of the human urinary bladder respond modestly to PHOTOFRIN (PII) PDT. Hypericum perforatum L., (St. John’s wort /Epirus’ Vasalmo, Greece), a medicinal plant used for many human ailments, is under investigation as a new photosensitizer. We have reported on the antiproliferative activity of the lipophilic extract of the Hypericum perforatum L. (HP) against cultured T-24, and NBT-11 bladder cancer cells. We investigated response of the polar methanolic fraction (PMF) of the HP extract versus PHOTOFRIN in photodynamic therapy (PDT) of human bladder cancer cells, RT-4 and T-24.The PMF was extracted from the dry herb with methanol, followed by liquid extraction with petroleum ether. RT-4/T-24, were plated (10⁵ cells/well) and placed in the incubator (37⁰ C, 5%CO) for 24 hours prior to addition of drugs. PII 2ug/ml, or PMF 60ug /ml was added and incubation continued. After 24 hours, the cells were treated with laser light (630nm) with 0,1,2,4 and 8 Joules. The cells were then washed and reincubated for another 24 hours. After this incubation cell survival was assessed by the MTT assay. PMF-PDT induced percent cell kill of 0%, 0%, 0%, 29% and 75%, in RT-4 cells (primary noninvasive urinary bladder TCC) versus 5%, 9%, 13%, 69% and 86%, in T-24 cells(metastatic TTC) at 0,1,2,4 and 8 Joules respectively. PII-PDT induced cell kill of 0 %, 0% ,0%,0% and 9 %, in RT-4 cells versus 0%,10%,0%,21% and 77%, in T-24 cells at 0,1,2,4 and 8 Joules respectively.RT-24 cells were relatively more resistant than T-24 cells to PMF and PII-PDT. Understanding mechanisms of such differential responses might prove useful

Photodynamic therapy (PDT) mediated with a vascular acting photosensitizer Tookad (pd-bacteriopheophorbide), was investigated as an alternative treatment modality for prostate cancer. Tookad photodynamic effects on the prostate and its adjacent tissues were evaluated in canine models. Interstitial prostate PDT was performed by irradiating individual lobes with a diode laser (763 nm) and 1-cm cylindrical diffuser fibers at various light doses to activate the IV administered photosensitizer Tookad (1 - 2 mg/kg). The sensitivity of the adjacent tissues to Tookad-PDT was determined by superficially irradiating the surfaces of the bladder, colon, abdominal muscle and pelvic plexus with a microlens fiber at various drug/light doses. PDT effect on the prostatic urethra was evaluated by transurethral irradiation. The prostate and adjacent tissues were harvested one-week after the treatment and subjected to histopathologic examination. At one-week post interstitial prostate PDT, the animals recovered well with little or no urethral complications. PDT induced prostate lesions were characterized by marked hemorrhagic necrosis. The bladder, colon, abdominal muscle and pelvic plexus, appeared to also be sensitive to Tookad-PDT at light dose levels greater than 40 Jcm². Urethral mucosa appeared less sensitive to Tookad-PDT. In conclusion, Tookad-mediated PDT demonstrates very strong vascular effects and can provide an effective alternative for the treatment of localized prostate cancer. Protection of the adjacent tissues should be taken into consideration in the total prostate ablation process due to their sensitivity to the Tookad-mediated PDT.

Photodynamic therapy of solid organs requires sufficient PDT dose throughout the target tissue while minimizing the dose to proximal normal structures. This requires treatment planning for position and power of the multiple delivery channels, complemented by on-line monitoring during treatment of light delivery, drug concentration and oxygen levels. We describe our experience in implementing this approach in Phase I/II clinical trials of the Pd-bacteriophephorbide photosensitizer TOOKAD (WST09)-mediated PDT of recurrent prostate cancer following radiation failure. We present several techniques for delivery and monitoring of photodynamic therapy, including beam splitters for light delivery to multiple delivery fibers, multi-channel light dosimetry devices for monitoring the fluence rate in the prostate and surrounding organs, methods of measuring the tissue optical properties in situ, and optical spectroscopy for monitoring drug pharmacokinetics of TOOKAD in whole blood samples and in situ in the prostate. Since TOOKAD is a vascular-targeted agent, the design and implementation of the techniques are different than for cellular-targeted agents. Further development of these delivery and monitoring techniques will permit full on-line monitoring of the treatment that will enable real-time, patient-specific and optimized delivery of PDT.

One of the most promising clinical applications of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PP IX) is the fluorescence diagnosis and photodynamic treatment of malignant brain tumours. In order to obtain a deeper understanding of the cellular processes involved, U373-MG human glioblastoma cells were used as model system to study intracellular location, fluorescence properties and light-induced reactions of the photosensitizer. In comparison with ovarian cancer or breast cancer cell lines, glioblastoma cells accumulated PP IX only to a moderate extent, but were most effectively inactivated (highest photodynamic efficacy per photosensitizer molecule). In contrast to breast cancer cells, which showed a rather granular PP IX fluorescence pattern, PP IX fluorescence in U373-MG cells seemed to originate mainly from cellular membranes. In addition, photobleaching and lowering of fluorescence lifetimes during irradiation were comparably small for this cell line. Combining the results of fluorescence lifetime imaging microscopy (FLIM) and photobleaching measurements, we deduced that those cellular sites where PP IX fluorescence was most pronounced, contributed rather little to its photodynamic efficacy.

Photodynamic therapy for the treatment of cancer relies on the presence of light, sensitizer and oxygen. By monitoring these three parameters during the treatment a better understanding and treatment control could possibly be achieved. Here we present data from in vivo treatments of solid skin tumors using an instrument for interstitial photodynamic therapy with integrated dosimetric monitoring. By using intra-tumoral ALA-administration and interstitial light delivery solid tumors are targeted. The same fibers are used for measuring the fluence rate at the treatment wavelength, the sensitizer fluorescence and the local blood oxygen saturation during the treatment. The data presented is based on 10 treatments in 8 patients with thick basal cell carcinomas. The fluence rate measurements at 635 nm indicate a major treatment induced absorption increase, leading to a limited light penetration at the treatment wavelength. This leads to a far from optimal treatment since the absorption increase prevents peripheral tumor regions from being fully treated. An interactive treatment has been implemented assisting the physician in delivering the correct light dose. The absorption increase can be compensated for by either prolonging the treatment time or increasing the output power of each individual treatment fiber. The other parameters of importance, i.e. the sensitizer fluorescence at 705 nm and the local blood oxygen saturation, are monitored in order to get an estimate of the amount of photobleaching and oxygen consumption. Based on the oxygen saturation signal, a fractionized irradiation can be introduced in order to allow for a re-oxygenation of the tissue.

Photodynamic therapy (PDT) has developed into a mature technology over the past several years, and is currently being exploited for the treatment of a variety of cancerous tumors, and more recently for age-related wet macular degeneration of the eye. However, there are still some unresolved problems with PDT that are retarding a more general acceptance in clinical settings, and thus, for the most part, the treatment of most cancerous rumors still involves some combination of invasive surgery, chemotherapy and radiation treatment, particularly subcutaneous tumors. Currently approved PDT agents are activated in the Visible portion of the spectrum below 700 nm, Laser light in this spectral region cannot penetrate the skin more than a few millimeters, and it would be more desirable if PDT could be initiated deep in the Near-infrared (NIR) in the tissue transparency window (700-1000 nm). MPA Technologies, Inc. and Rasiris, Inc. have been co-developing new porphyrin PDT designed to have greatly enhanced intrinsic two-photon cross-sections (>800 GM units) whose two-photon absorption maxima lie deep in the tissue transparency window (ca. 780-850 nm), and have solubility characteristics that would allow for direct IV injection into animal models. Classical PDT also suffers from the lengthy time necessary for accumulation at the tumor site, a relative lack of discrimination between healthy and diseased tissue, particularly at the tumor margins, and difficulty in clearing from the system in a reasonable amount of time post-PDT. We have recently discovered a new design paradigm for the delivery of our two-photon activated PDT agents by incorporating the porphyrins into a triad ensemble that includes a small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR one-photon imaging agent that allows the tracking of the triad in terms of accumulation and clearance rates. We are currently using these new two-photon PDT triads in efficacy studies with two breast cancer cell lines, both in vitro and in vivo. Both of these cell lines have been transfected with luciferase genes that allow implanted tumor growth and PDT efficacy to be monitored in living mouse models over time by following the rise and decay of the bioluminescence signals.

This report focuses on the treatment parameters leading to successful nanoshell-assisted photo-thermal therapy (NAPT). NAPT takes advantage of the strong near infrared (NIR) absorption of gold-silica nanoshells, a new class of nanoparticles with tunable optical absorptivities that are capable of passive extravasation from the abnormal tumor vasculature due to their nanoscale size. Under controlled conditions nanoshells accumulate in tumors with superior efficiency compared to surrounding tissues. For this treatment: (1) tumors were inoculated in immune-competent mice by subcutaneous injection, (2) polyethylene glycol coated nanoshells (≈150 nm diameter) with peak optical absorption in the NIR were intravenously injected and allowed to circulate for 6 - 48 hours, and (3) tumors were then extracorporeally illuminated with a collimated diode laser (808 nm, 2-6 W/cm2, 2-4 min). Nanoshell accumulations were quantitatively assessed in tumors and surrounding tissues using neutron activation analysis for gold. In order to assess temperature elevation, laser therapies were monitored in real-time using a mid-infrared thermal sensor. NAPT resulted in complete tumor regression in >90% of the subjects. This simple, non-invasive procedure shows great promise as a technique for selective photo-thermal tumor treatment.

The photodynamic action of the Hypericum perforatum L. extract, mainly its polar methanolic fraction (PMF) has recently been substantiated by our group. The herb contains a number of naphthodianthrones - photosensitizers mainly hypericin and pseudohypericin. The concentration of hypericins in PMF was found to be 1.37 %. The distribution of hypericins fluorescence in sections of normal rat bladder tissues after the intravesical instillation of the polar methanolic fraction of hypericum (PMF) was studied by the use of fluorescence microscopy. PMF was dissolved in normal saline containing 0.5 μg/ml concentration of hypericins, and was then instilled in rat bladder for 15, 30, 60 and 120 minutes respectively. PMF solutions were withdrawn, bladders were rinsed through the catheter with normal saline and rats were sacrificed. Bladders were then removed, cut open and immediately mounted in medium, and immersed in liquid nitrogen. Two consecutive 3-μm frozen sections were cut with a cryostat. The first section was examined by fluorescence microscopy and the second section was stained with hematoxylin and eosin. For fluorescence imaging the filter set used included a 535/50 nm bandpass excitation filter and a 610/75 nm emission filter. Fluorescence images were acquired and documented using photography. Fluorescene could be detected in bladder samples after only 15 minutes of instillation with the above described solution. The urothelium / muscle fluorescence ratio ranged from 5/1 to 11/1 in various sites of the samples examined. No fluorescence originating from the muscle could be detected. PMF should be further studied towards the direction of its use in photodynamic therapy.

Time-resolved photoacoustic imaging has been used to characterize Breast tissues for the purpose of discriminating between normal and Cancerous tumor areas of tissue. Ultrasonic thermoelastic waves were generated in Breast tissue by the absorption of nanosecond laser pulses at 193 nm produced by a frequency doubled Q-switched excimer laser in conjunction with an optical interferometer sensor was used to detect the thermoelastic and thermal waves. At 193 nm, differences in photoacoustic and photothermal signatures of normal tissue and Cancerous tumor were found to be highly enhanced. There was a clear and reproducible difference between the photacoustic and photothermal response of Cancerous tumor and normal tissue as a result of increased optical attenuation in Cancerous tumor. At 193 nm, the generation of subsurface thermoelastic waves provided a means of determining the structure and thickness of the tissue sample. The thermal waves provided a mean of determination the optical absorption of the tissue sample.

Isotropic detectors with spherical scattering tips are commonly used for in-vivo dosimetry of light fluence rate during photodynamic therapy (PDT). These detectors are typically calibrated in-air. It has been well established that the response of an isotropic detector is a function of the refractive index (n) of the surrounding medium when it is surrounded by an infinite medium of uniform n. However, there are few, if any, studies of the isotropic detector response when the detector is placed in a secondary medium, such as air, before it is placed inside the infinite uniform medium. This condition often arises when one places the isotropic detector inside an air-filled catheter which is then inserted into a turbid medium, such as tissue. We have performed theoretical and experimental studies to determine the correction factors in water (n = 1.33), which has a refractive index similar to that of tissue (n = 1.4). We found that the resulting correction factor is almost the same (within 20%) as the correction factor for the outermost medium (the water) rather than the immediate medium surrounding the isotropic detector (air). The detector correction factor is also a function of the index of refraction of the probe material. For a 1-mm diameter probe from CardioFocus, the detector correction factor varied from 1 (in air) to 1.09 (at air-water interface) to 1.49 (completely submerged in water). At the air-water interface the spherical bulb of the isotropic detector is placed half in air and half in water. For a 0.5-mm diameter probe from the same company, it varied from 1 (in air) to 1.32 (at air-water interface) to 1.87 (in water). For a 0.3-mm diameter probe from the same company, it varied from 1 (in air) to 1.32 (at air-water interface) to 1.71 (in water). We have also found that the detector response changes by less than 10% when the detector position is varied from touching the catheter wall closest to the light source, to not touching, to touching the catheter wall farthest from the light source. The calibration factors between individual isotropic detectors of the same type varied within 5% for all detector types. Thus mean correction factor can be used for each individual isotropic detector of the same type.

To deliver uniform photodynamic dose to the prostate gland, it is necessary to develop algorithms that optimize the location and strength (emitted power × illumination time) of each light source. Since tissue optical properties may change with time, rapid (almost real-time) optimization is desirable. We use the Cimmino algorithm because it is fast, linear, and always converges reliably. A phase I motexafin lutetium (MLu)-mediated photodynamic therapy (PDT) protocol is on-going at the University of Pennsylvania. The standard plan for the protocol uses equal source strength and equal spaced loading (1-cm). PDT for the prostate is performed with cylindrical diffusing fibers (CDF) of various lengths inserted to longitudinal coverage within the matrix of parallel catheters perpendicular to a base plate. We developed several search procedures to aid the user in choosing the positions, lengths, and intensities of the CDFs. The Cimmino algorithm is used in these procedures to optimize the strengths of the light catheters at each step of the iterative selection process. Maximum and minimum bounds on allowed doses to points in four volumes (prostate, urethra, rectum, and background) constrain the solutions for the strengths of the linear light sources. Uniform optical properties are assumed. To study how different opacities of the prostate would affect optimization, optical kernels of different light penetration were used. Another goal is to see whether the urethra and rectum can be spared, with minimal effect on PTV treatment delivery, by manipulating light illumination times of the sources. Importance weights are chosen beforehand for organ volumes, and normalized. Compared with the standard plan, our algorithm is shown to produce a plan that better spares the urethra and rectum and is very fast. Thus the combined selection of positions, lengths, and strengths of interstitial light sources improves outcome.

To elucidate the mechanism of photosensitization with pulsed light excitation, we previously introduced fluorescence-oxygen diagram that shows the correlation between photochemical oxygen consumption and photobleaching during a treatment (Kawauchi et al., Photochem. Photobiol., 80, 216-223, 2004). In pulsed photodynamic treatment of A549 cells with ATX-S10•Na(II), the diagrams for treatments at relatively high repetition rates of 10 and 30 Hz showed the complex behaviors of photochemical reaction; photobleaching initially occurred with oxygen consumption but it was switched to oxygen-independent photobleaching, which was followed by a secondary oxygen-consuming regime. In this study, fluorescence microscopy revealed that for treatments at 10 and 30 Hz, subcellular fluorescence distribution of ATX-S10•Na(II) changed drastically from the high-intensity spotty patterns showing lysosomal accumulation to the diffusive patterns within the cytosol during certain ranges of total light dose. These ranges were found to coincide with those in which oxygen-independent reaction appeared. These findings suggest that the sensitizer started to be redistributed from lysosomes to the cytosol during the oxygen-independent reaction regime. On the other hand, at 5 Hz, such reaction switching was not clearly seen during whole irradiation period in the diagram; this was consistent with the observation that sensitizer redistribution efficiently occurred even in the early phase of irradiation. The appearance of oxygen-independent reaction at the higher repetition rates may be caused by high local concentration of the sensitizer and the resultant low concentration of oxygen in the reaction sites due to the shorter pulse-to-pulse time intervals. In pulsed photodynamic treatment, pulse frequency is an important parameter that affects the intracellular kinetics of the sensitizer and hence the photochemical reaction dynamics.

Photodynamic activity of several dyes on Drosophila melanogaster at different temperatures (15-35°C) inside of test-tubes was investigated. Both phototoxic sensitizers (chlorin e6, methylene blue, etc. -group A) and non active compounds (hemoglobin, brilliant green, pyronine, etc.-group B) were used. Dyes of 10^-5-10^-3 M concentration were added to the food for drosophila 24 hours before irradiation. Solar radiation, narrow-band halogen lamps, LEDs and laser were used as a photo-stimulator. Irradiation parameters: I ≤ 45mW/cm² and 0.2<t_irr<1.5 h. Without using dyes in the control test-tubes at all doses of irradiation and temperatures applied percentage of survived insects was approximately 100%. In the darkness with the use of all dyes observations also indicated no damage to the insects. At the temperatures up to 25°C when using dyes of group B insects were not affected at all, while with the dyes of group A findings showed dose-dependent insect mortality. At high temperatures (30-35°C) when using group B dyes flies were losing their mobility and in the case of group A dyes the drosophila’s survival value sharply dropped. Combination of dyes from A group with some dyes from B group leads to the partial disappearance of photodynamic effect. This, probably, is concerned with the toxic photoproduct suppression by the inactive dye. Experimental model of drosophila allows to investigate photosensitization impact within wide temperature range, to find out the processes, when using combination of dyes, as well as to study photodynamic effect on reproductive functions of insects.

Fluorescence diagnosis of malignant lesions has been showed as an attractive optical technique due especially to its real-time response and a more objective and quantitative evaluation. Even though the oral cavity allows a direct examination many lesions are diagnosed when it is already in advanced stage, compromising the patient prognosis. In this study, the fluorescence spectroscopy was used to the detection of chemically induced carcinoma at the lateral border of the tongue in a hamster model. Two excitations wavelengths in visible region were applied: 442 and 532 nm. All the spectra results were analyzed comparing with the histopathological diagnosis. The better results were achieved with the 442 nm laser excitation. The spectra from carcinoma showed new emission bands and these were used to determined different ratios for a quantitative analysis. Using the 625-645 nm fluorescence range under 442 nm excitation (A₃ coefficient) the percentage of false negative was of 9.1%, however the false positive percentage was of 18.5%. The 532 nm excitation provided a better normal tissue detection compared to 442 nm excitation. The ideal clinical condition is probably the use of multiple wavelengths excitation for a broader tissue fluorescence investigation.

Clinical application of Photodynamic Therapy (PDT) in Brazil is a result of a pioneering work in a collaborative program involving the Physics Institute and the Medical School of the University of Sao Paulo and the Amaral Carvalho Cancer Hospital in the city of Jau, Sao Paulo. This work began in 1997 with the first patient treated in 1999. Up to the end of 2003 this program has treated over 300 patients and the ones with correct follow up had their lesions included in this report. The majority of the lesions were of non-melanoma skin cancer located on the head and neck region, but the group has also treated Esophagus, Bladder, Gynecological, chest wall recurrence of breast cancer, among others. The results have shown to be compatible with internationally reported data, and we have modified some application procedures towards to a better benefit for the patient and an optimization of the results. We present the overall results observed after 5 year of experimental clinical treatment.

We have performed an experiment to investigate the degradation of the photosensitizer Photogem in solution. The investigation was carried out at 630 nm diode laser for different intensities. The light degradation considered was from light that could cause modification of the solution fluorescence spectra under excitation of 535 nm. Knowing the degradation rate as a function of intensity, one can infer the rate of degradation during PDT application and its process efficiency loss.

The endogenous photosensitizer 5-aminolevulinic acid (ALA) is a haem precursor and induces the synthesis of protoporphyrin IX (PpIX) in mitochondria-containing cells. Due to the slow conversion of porphyrins to haem, high levels of PPIX are found in the tissues, sufficient to produce a photodynamic effect following exposure to light. Since PpIX accumulates effectively in tumor cells, the use of ALA leads to a better photoselectivity than Photofrin. However, this selectivity has not been sufficiently studied. As far as we know there is just one study comparing the amount of accumulated PpIX in non-tumor and tumor cell lines. In this work we attempt to compare not just the production but also the accumulation and cytotoxicity of PpIX in non-tumor (VERO) versus tumor (Hep-2) cells induced by the use of ALA. The results have shown that both non-tumor and tumor cell lines produce the same amount of PpIX but just the tumor cells can accumulate PpIX. So, under illumination, only the tumor cells will be killed.

Photodynamic therapy (PDT) is a technique for inducing tissue damage with light irradiation of a drug selectively retained in malignant tissue in presence of oxygen. The same mechanisms responsible for PDT efficacy can destroy the sensitizer, a process called photobleaching. In this work, the photobleaching of Photogem (a hematoporphyrin derivative used in Brazil made in Russia) was induced to study phototoxicity of the photoproducts upon a tumor (HEp-2) and non-tumor (VERO) cell line. Photogem was previously irradiated at 514nm and 630 nm (50mW/cm²) for 120 min. The sensitizer photobleaching was monitored by fluorescence and absorption properties changes and photoproducts formation evidenced by the appearance of a new absorption band around 640nm. Irradiated and non-irradiated Photogem were incubated for 18h with the cells. After drug removal, cells were irradiated with LED at 630 nm with intensities of 13, 20 and 25mW/cm² for different times. Then, the cells were incubated for 48 hours to determine the cells viability by the MTT method. The cells in the dark were used as control. The IC50 decreases as light intensity increases, being more pronounced for tumor cells. Previously irradiated Photogem needs one-hour irradiation for both cell lines to have the same IC50 value of non-irradiated Photogem, which are irradiated for 14 min in tumor cells and 25 min in non-tumor cells. The results suggest that the photoproducts of Photogem are less cytotoxic either in the dark or in the light, decreasing with increase of intensity. These results underline the importance of dosimetry in PDT.

In this paper, we optimized the PDT laser system to improve the therapy effects of malignancies. In order to optimizes, the variation of laser output and specific wavelength shift have to reduced. To improved the PDT therapy clincian require the diverse radiation mode which irradiate the tumor surface. Continuous wave mode that general application may causes tissue thermal damage not only to tumor tissue, but also to nomal tissue. Therefore, we suggested new technique for radiation method to improved PDT effects and prevented to the thermal effects for the tissue. In experimental we verified the stability of wavelength, laser output stability and proved the reduced thermal effects to the tissue using the pulse & burst radiation modes in vitro.

The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the α_vβ₃ integrin was assessed from xenografts bearing a subcutaneous human Kaposi’s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi’s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

We previously demonstrated the methylene blue-mediated photodynamic inactivation of P. aeruginosa in vitro using pulsed light excitation. However, the photobactericidal mechanism has not been revealed yet. In this study, morphological changes induced by pulsed photodynamic treatment in bacteria were investigated by electron microscopy. The micrographs of bacteria showed an alteration in the membrane structure in the early period of illumination, and increase in the total light dose caused an alteration in the cytoplasmic structure. It was shown that the membrane damage may be lethal to bacteria.

The necessity of researches of antitumor efficiency of new photosensitizers (PS) is explained by the opportunity of their application in photodynamic therapy of tumors. PS, selectively accumulated in cancer cells and activated by the light, generate the active oxygen species that cause apoptosis. Earlier, it was shown that PS chlorin e6 (0.3-0.5 μg/ml) induces rat embryo fibroblast-like cell apoptosis. In present work antitumor activity of the new photosensitizers, water-soluble cationic porphyrins and their metal complexes, is investigated. The dose-dependent destruction of cancer cells was shown on PC-12 (pheochromocytoma, rat adrenal gland) and Jurkat (human lymphoma) cell lines. Meso-tetra-[4-N-(2 `- oxyethyl) pyridyl] porphyrin (TOEPyP) and chlorin e6 possessed the same toxicity at LD50 dose on PC-12 cell line, whereas phototoxicity of TOEPyP was 3 times less compared to chlorin e₆(LD₅₀=0.2 and 0.075 μg/ml accordingly). The results have shown weak photosensitizing effect of Zn-and Ag-derivatives of TOEPyP on PC-12 cell line. TOEPyP and Zn-TOEPyP (0.1 - 50 μg/ml) were non-toxic for Jurkat cell line, whereas Ag-TOEPyP was toxic at 10 μg/ml (LD₉₀). TOEPyP and chlorin e₆ have shown phototoxic effect in the same dose range (LD₅₀=0.5 and 0.3 μg/ml accordingly). The investigation of toxic and phototoxic effects of the new porphyrins revealed significantly different sensitivity of various cell lines to PSs.

Hypericin is a novel, highly fluorescent photosensitizer that exhibits selective tumor cell uptake properties and is particularly resistant to photobleaching. In this study, we have characterized hypericin uptake in human pancreatic tumor cells with relation to incubation time, cell number, and drug concentration. Ex vivo hypericin based fluorescence spectroscopy was performed to detect the presence of MIA PaCa-2 pancreatic tumor cells in the peritoneal cavity of BALB/c nude mice, as well as to quantify gross tumor burden. Hypericin based cytology of peritoneal lavage samples, using both one and two photon laser confocal microscopy, demonstrated more than a two-fold increase in fluorescence emission of pancreatic tumor cells as compared to control samples. In vitro treatment of pancreatic cancer cells with hypericin based photodynamic therapy showed tumor cell cytotoxicity in a drug dose, incident laser power, and time dependent manner. For these experiments, a continuous wavelength solid-state laser source (532 nm) was operated at power levels in the range of 100-400 mW. Potential applications of hypericin in tumor diagnosis, staging, and therapy will be presented.

The photodynamic therapy with saturating of photochemical dose (PD) using three different techniques was studied theoretically. At saturation the PD value does not depend on the light fluence and is determined by concentration of photosensitizer or oxygen. The possibility of creating more uniform therapeutic dose distribution throughout a tumor is shown.

Among the challenges to the clinical implementation of photodynamic therapy (PDT) is the delivery of a uniform photodynamic dose to induce uniform damage to the target tissue. As the photodynamic dose depends on both the local sensitizer concentration and the local fluence rate of treatment light, knowledge of both of these factors is essential to the delivery of uniform dose. In this paper, we investigate the distribution and kinetics of the photosensitizer motexafin lutetium (MLu, Lutrin) as revealed by its fluorescence emission. Our current prostate treatment protocol involves interstitial illumination of the organ via cylindrical diffusing fibers (CDF’s) inserted into the prostate though clear catheters. For planning and treatment purposes, the prostate is divided into 4 quadrants. We use one catheter in each quadrant to place an optical fiber-based fluorescence probe into the prostate. This fiber is terminated in a beveled tip, allowing it to deliver and collect light perpendicular to the fiber axis. Excitation light is provided by a 465 nm light emitting diode (LED) source coupled to a dichroic beamsplitter, which passes the collected fluorescence emission to a CCD spectrograph. Spectra are obtained before and after PDT treatment in each quadrant of the prostate and are analyzed via a linear fitting algorithm to separate the MLu fluorescence from the background fluorescence originating in the plastic catheter. A computer-controlled step motor allows the excitation/detection fiber to be moved along the catheter, building up a linear profile of the fluorescence emission spectrum of the tissue as a function of position. We have analyzed spectral fluorescence profiles obtained in 4 patients before and after MLu-mediated PDT. We find significant variation both within individual prostates and among patients. Within a single quadrant, we have observed the fluorescence signal to change by as much as a factor of 3 over a distance of 2 cm. Comparisons of pre- and post-PDT spectra allow a quantification treatment-induced photobleaching. Like the drug distribution, the extent of photobleaching varies widely among patients. In two cases, we observed bleaching of approximately 50% of the drug, while others exhibited negligible photobleaching.

Photodynamic therapy against murine macrophage like cells with the second-generation hydrophilic photosensitizer ME2906 (mono-L-aspartyl chlorin e₆:NPe6) was performed in vitro to study therapeutic effect distribution formation along depth with high-intensity pulsed irradiation. The photocytotoxicity of macrophage like cell with ME2906 under various fluence rates was measured. We found photocytotoxicity suppression from 64% to 16% in the cell lethality ranging the fluence rate of a pulsed laser from 20 to 400mW/cm² (corresponding pulse peak power: from 0.07 to 1.4 MW/cm²). The cell lethality of about 80 % was obtained with continues wave laser irradiation under the fluence of 10 J/cm². Photobleaching and oxygen consumption of the photosensitizer solution, were measured to know photoreaction of the photosensitizer solution under the high fluence rate pulsed irradiation. Type-II photochemical reaction suppression was indicated with the high fluence rate pulsed irradiation. The transient absorption of the photosensitizer solution during pulse irradiation was measured by the pump-and-probe technique with pulse peak power density up to 1.2 MW/cm² to investigate absorption saturation. In the case of the pump beam peak power of 1.2MW/cm², the transmittance of the probe beam increased approximately 7% from that of without the pump beam, so that huge absorption saturation did not occur in this case. We think the main cause of the photocytotoxicity suppression in this study may not to be attributed to the absorption saturation. This photocytotoxicity suppression induced by the high-intensity irradiation may be available to control treatment depth of PDT to preserve healthy internal wall of a hollow organ.

During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.