This PDF file contains the front matter associated with SPIE Proceedings Volume 7164, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

While the first reactive oxygen species (ROS) formed during photodynamic therapy (PDT) is singlet molecular oxygen (¹O₂), other ROS are formed downstream including superoxide anion radical (•CO₂ ^-), hydrogen peroxide (H₂O₂) and hydroxyl radical (•OH). In this study, we examined the role of H₂O₂ in the phototoxic response to PDT in murine leukemia L388 cells. Inhibition of catalase activity, a major pathway to H₂O₂ detoxification, led to enhanced apoptosis and cell death. Addition of exogenous catalase offered protection from phototoxicity as did chelation of Fe⁺², a co-factor in •OH production from H₂O₂. These results indicate the H₂O₂ formed during PDT plays a role in PDT efficacy.

Autophagy (or more properly, macroautophagy) is a pathway whereby damaged organelles or other cell components are encased in a double membrane, the autophagosome, which fuses with lysosomes for digestion by lysosomal hydrolases. This process can promote cell survival by removing damaged organelles, but when damage is extensive, it can also be a mechanism of cell death. Similar to the Kessel and Agostinis laboratories, we have reported the vigorous induction of autophagy by PDT; this was found in human breast cancer MCF-7 cells whether or not they were able to efficiently induce apoptosis. One way to evaluate the role of autophagy in PDT-treated cells is to silence one of the essential genes in the pathway. Kessel and Reiners silenced the Atg7 gene of murine leukemia L1210 cells using inhibitory RNA and found sensitization to PDT-induced cell death at a low dose of PDT, implying that autophagy is protective when PDT damage is modest. We have examined the role of autophagy in an epithelium-derived cancer cell by comparing parental and Atg7-silenced MCF-7 cells to varying doses of PDT with the phthalocyanine photosensitizer Pc 4. In contrast to L1210 cells, autophagy-deficient MCF-7 cells were more resistant to the lethal effects of PDT, as judged by clonogenic assays. A possible explanation for the difference in outcome for L1210 vs. MCF-7 cells is the greatly reduced ability of the latter to undergo apoptosis, a deficiency that may convert autophagy into a cell-death process even at low PDT doses. Experiments to investigate the mechanism(s) responsible are in process.

We constructed a whole-body fluorescence tomography instrument to monitor novel bifunctional phototherapeutic drugs (e.g., HPPH-Cyanine dye conjugate) in small animals. The instrument allows dense source and detector sampling with a fast galvo scanner and a CCD detector for improved resolution and sensitivity (Patwardhan et al., 2005). Here we report tissue phantom measurements to evaluate the imaging performance with a newly constructed tomography instrument. Phantom measurements showed that strong fluorescence generated by HPPH-Cyanine dye (HPPH-CD), having high fluorescence quantum yield and long wavelength fluorescence emission, allowed deep tissue imaging. We also report in vivo fluorescence measurements of the conjugate in Nude mice bearing A549 human non-small cell lung carcinoma (NSCLC) tumors at 24 hr post injection to evaluate tumor detection ability of the conjugate. Our results indicate that the HPPH-CD shows preferential uptake in tumors compared to surrounding normal tissue at 24 hr post injection. This study demonstrates a potential use of HPPH-CD in detection (fluorescence imaging) and treatment (PDT) of deeply seated tumors.

In-vivo light Dosimetry for patients undergoing photodynamic therapy (PDT) is one of the important dosimetry quantities critical for predicting PDT outcome. This study examines the light fluence (rate) delivered to patients undergoing pleural PDT as a function of treatment time, treatment volume and surface area, and its accuracy as a function of the calibration accuracies of each isotropic detector and the calibration integrating sphere. The patients studied here were enrolled in Phase II clinical trial of Photofrin-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. The ages of the patients studied varied from 34 to 69 year old. All patients were administered 2mg per kg body weight Photoprin 24 hours before the surgery. Patients undergoing photodynamic therapy (PDT) are treated with laser light with a light fluence of 60 J/cm^2 at 630nm. Fluence rate (mW/cm^2) and cumulative fluence (J/cm^2) was monitored at 7 different sites during the entire light treatment delivery. Isotropic detectors were used for in-vivo light dosimetry. The anisotropy of each isotropic detector was found to be within 30%. The mean fluence rate delivery varied from 37.84 to 94.05 mW/cm^2 and treatment time varied from 1762 to 5232s. We have established a correlation between the treatment time and the treatment volume. The results are discussed using an integrating sphere theory and the measured tissue optical properties. The result can be used as a clinical guideline for future pleural PDT treatment.

The object of this study is to develop optimization procedures that account for both the optical heterogeneity as well as photosensitizer (PS) drug distribution of the patient prostate and thereby enable delivery of uniform photodynamic dose to that gland. We use the heterogeneous optical properties measured for a patient prostate to calculate a light fluence kernel (table). PS distribution is then multiplied with the light fluence kernel to form the PDT dose kernel. The Cimmino feasibility algorithm, which is fast, linear, and always converges reliably, is applied as a search tool to choose the weights of the light sources to optimize PDT dose. Maximum and minimum PDT dose limits chosen for sample points in the prostate constrain the solution for the source strengths of the cylindrical diffuser fibers (CDF). We tested the Cimmino optimization procedures using the light fluence kernel generated for heterogeneous optical properties, and compared the optimized treatment plans with those obtained using homogeneous optical properties. To study how different photosensitizer distributions in the prostate affect optimization, comparisons of light fluence rate and PDT dose distributions were made with three distributions of photosensitizer: uniform, linear spatial distribution, and the measured PS distribution. The study shows that optimization of individual light source positions and intensities are feasible for the heterogeneous prostate during PDT.

Pancreatic cancer generally has very poor prognosis, with less than 4% survival at 5 years after diagnosis. This dismal survival rate is in part due to the aggressive nature of the adenocarcinoma, leading to a late-stage at diagnosis and exhibits resistance to most therapies. Photodynamic therapy (PDT) is a model cellular and vascular therapy agent, which uses light activation of the delivered drug to photosensitize the local cellular millieu. We suggest that interstitial verteporfin (benzoporphyrin derivative monoacid ring A) PDT has the potential to be an adjuvant therapy to the commonly used Gemcitabine chemotherapy. In the current study, an orthotopic pancreatic cancer model (Panc-1) has undergone interstitial verteporfin PDT (40 J/cm with verteporfin and 40 J/cm without verteporfin). Prior to PDT, magnetic resonance (MR) imaging was used to determine the location and size of the tumor within the pancreas, allowing accurate placement of the diffusing fiber. The success of therapy was monitored in vivo by assessing the total tumor and vascular perfusion volumes 24 hours pre- and 48 hours post-PDT. Total tumor and vascular perfusion volumes were determined using T2 weighted (T2W) and Gd-DTPA difference T1 weighted (T1W) turbo spin echo (TSE) MR imaging sequences, respectively. The validity of the in vivo imaging for therapeutic response was confirmed by ex vivo fluorescence and histological staining of frozen tissue sections. The ex vivo DiOC7(3) fluorescence analysis correlates well with the information provided from the MR images, indicating that MR imaging will be a successful surrogate marker for interstitial PDT.

Intracellular accumulation and location of photosensitizers, e.g. 5-ALA induced protoporphyrin IX, are crucial parameters for the efficiency of photodynamic therapy (PDT). Fluorescence microscopy has proved to be a powerful technique to assess these parameters, however, even at low light doses around or below 1 J/cm² cells may be irreversibly damaged. Therefore, prior to microscopic experiments non-phototoxic light doses were determined, and experimental conditions of laser scanning (LSM) and wide field microscopy were adapted to these doses. Wide field images appeared more brilliant than LSM images, thus demonstrating some advantage of simultaneous over sequential detection. In addition, human glioblastoma cells appeared less sensitive towards illumination by an evanescent electromagnetic field than towards epi-illumination, since only their plasma membranes and adjacent parts were exposed to light.

Patients with non-resectable pancreatic and biliary tract cancer (cholangiocarcinoma and gallbladder cancer) have a dismal outlook with conventional palliative therapies, with a median survival of 3-9 months and a 5 year survival of less than 3%. Surgery is the only curative treatment but is appropriate in less than 20% of cases, and even then is associated with a 5-year survival of less than 30%. Although most applications of photodynamic therapy (PDT) in gastroenterology have been on lesions of the luminal gut, there is increasing experimental and clinical evidence for its efficacy in cancers of the pancreas and biliary tract. Our group has carried out the only clinical study of PDT in pancreatic carcinoma reported to date, and showed that PDT is feasible for local debulking of pancreatic cancer. PDT has also been used with palliative intent in patients with unresectable cholangiocarcinoma, with patients treated with stenting plus PDT reporting improvements in cholestasis, quality of life and survival compared with historical or randomized controls treated with stenting alone. Further controlled studies are needed to establish the influence of PDT and chemotherapy on the survival and quality of life of patients with pancreatic and biliary tract carcinoma.

Nonmelanoma skin carcinomas are the most common of all human cancers. Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) has been used to treat these tumors, but has shown variable results. We are pursuing a multifaceted approach toward optimizing tumor responsiveness. First, a new paradigm is being developed in which tumors are pretreated with differentiation-inducing agents, e.g. methotrexate or Vitamin D, to enhance synthesis of protoporphyrin IX (PpIX) and improve tumor cell killing upon exposure to 635 nm light. This principle was first elucidated in cell culture studies, and has now been shown to hold true for murine skin tumors, and for a human subcutaneous tumor model (A431 cells injected in nude mice). Clinical trials to test methotrexate and Vitamin D as augmenting agents for ALA-PDT of nonmelanoma skin cancer are being designed. Second, better methods to measure PpIX in patients' skin tumors in real time are being developed. In a clinical study to measure PpIX in patients with dysplastic skin lesions, in vivo fluorescence dosimetry was used to measure the accumulation of PpIX over time, and revealed that intralesional PpIX may reach clinically-useful levels earlier than previously thought for the treatment of actinic keratoses. In a second clinical study to examine depth of PpIX production in nonmelanoma skin cancer, the depth of PpIX within BCC tumors was found at relatively deep levels (>1 mm) in some tumor nests, but not in others. Production of PpIX in deep squamous cell carcinoma was very low. In summary, molecular approaches such as differentiation therapy to enhance ALA-PDT for individual patients may ultimately be needed to help to improve skin cancer responses to this modality.

We present the methods that are being used in the scope of an on-going clinical trial designed to assess the usefulness of ALA-PpIX fluorescence imaging when used in conjunction with pre-operative MRI. The overall objective is to develop imaging-based neuronavigation approaches to aid in maximizing the completeness of brain tumor resection, thereby improving patient survival rate. In this paper we present the imaging methods that are used, emphasizing technical aspects relating to the fluorescence optical microscope, including initial validation approaches based on phantom and small-animal experiments. The surgical workflow is then described in detail based on a high-grade glioma resection we performed.

A system is presented which has been developed for dermatological applications with the need to quantify levels of protoporphyrin IX in diagnosis or therapy. The design was to couple fluorescence sampling onto a high frequency ultrasound system and take multiple optical source-detector samples of the tissue of the fluorescence and transmission signals. The intensity values can then be used to estimate and image the PpIX levels present in tissue samples. The system design, calibration, and initial testing in tissue phantoms are demonstrated here. The component design has been modular and allows easy implementation as a kit which can be assembled from basic components. The control software is more elaborate and provides a seamless way to go from system start up through to fluorescence quantification of PpIX concentrations.

Singlet oxygen (¹O₂) is generally believed to be the major cytotoxic agent during photodynamic therapy (PDT), and the reaction between ¹O₂ and tumor cells define the treatment efficacy. From a complete set of the macroscopic kinetic equations which describe the photochemical processes of PDT, we can express the reacted ¹O₂ concentration, [¹O₂]_rx, in a form related to time integration of the product of ¹O₂ quantum yield and the PDT dose rate. The production of [¹O₂]_rx involves physiological and photophysical parameters which need to be determined explicitly for the photosensitizer of interest. Once these parameters are determined, we expect the computed [¹O₂]_rx to be an explicit dosimetric indicator for clinical PDT. Incorporating the diffusion equation governing the light transport in turbid medium, the spatially and temporally-resolved [¹O₂]_rx described by the macroscopic kinetic equations can be numerically calculated. A sudden drop of the calculated [¹O₂]_rx along with the distance following the decrease of light fluence rate is observed. This suggests that a possible correlation between [¹O₂]_rx and necrosis boundary may occur in the tumor subject to PDT irradiation. In this study, we have theoretically examined the sensitivity of the physiological parameter from two clinical related conditions: (1) collimated light source on semi-infinite turbid medium and (2) linear light source in turbid medium. In order to accurately determine the parameter in a clinical relevant environment, the results of the computed [¹O₂]_rx are expected to be used to fit the experimentally-measured necrosis data obtained from an in vivo animal model.

An interstitial diffuse optical tomography (iDOT) system with multiple light diffusers and isotropic detectors has been developed to characterize the optical properties of prostate gland during photodynamic therapy (PDT). During the data acquisition, linear or point sources and detectors are inserted into the prostate gland, sequentially, and controlled by a motorized system. For our continuous-wave (CW) iDOT system, CW measurements of optical signal are made, and the spatial distributions of light fluence rate can be described by the CW diffusion equation. Optical properties (absorption and reduced scattering coefficients) of the prostate gland are reconstructed by solving the inverse problem with the use of an adjoint model based on the CW diffusion equation. To exam our methodology, two and three dimensional mathematical prostate phantoms including anomalies with known optical properties is prepared and we compare the absorption and reduced scattering images reconstructed for the phantom with the known results. In the end, we discuss the issue of reconstruction of optical properties using human patient data.

We present the results of a series of spectroscopic measurements made in vivo in patients undergoing photodynamic therapy (PDT). The patients studied here were enrolled in Phase II clinical trials of Photofrin-mediated PDT for the treatment of non-small cell lung cancer and cancers with pleural effusion. Patients were given Photofrin at dose of 2 mg per kg body weight 24 hours prior to treatment. Each patient received surgical debulking of the tumor followed by intracavity PDT at 630nm to a dose of 60 J/cm2. Dose was monitored continuously using implanted isotropic fiber-based light detectors. We measured the diffuse reflectance spectra before and after PDT in various positions within the cavity, including tumor, diaphragm, pericardium, skin, and chest wall muscle in 10 patients. The measurements were acquired using a specially designed fiber optic-based probe consisting of one fluorescence excitation fiber, one white light delivery fiber, and 9 detection fibers spaced at distances from 0.36 to 7.8 mm from the source, all of which are imaged via a spectrograph onto a CCD, allowing measurement of radially-resolved diffuse reflectance and fluorescence spectra. The absorption spectra were analyzed using an analytical model of light propagation in diffuse media based on the P3 approximation to radiative transport, assuming a known basis set of absorbers including hemoglobin in its oxygenated and deoxygenated forms and Photofrin. We find significant variation in hemodynamics and sensitizer concentration among patients and within tissues in a single patient.

Periodontitis affects half of the U.S. population over 50, and is the leading cause of tooth loss after 35. It is believed to be caused by growth of complex bacterial biofilms on the tooth surface below the gumline. Photodynamic therapy, a technology used commonly in antitumor applications, has more recently been shown to exhibit antimicrobial efficacy. We have demonstrated eradication of the periopathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans in vitro using Periowave^TM; a commercial photodisinfection system. In addition, several clinical studies have now demonstrated the efficacy of this treatment. A pilot study in the U.S. showed that 68% of patients treated with Periowave^TM adjunctively to scaling and root planing (SRP) showed clinical attachment level increase of >1 mm, as opposed to 30% with SRP alone. In a subsequent larger study, a second Periowave^TM treatment 6 weeks after initial treatment led to pocket depth improvements of >1.5 mm in 89% of patients. Finally, in the most recent multicenter, randomized, examiner-blinded study conducted on 121 subjects in Canada, Periowave^TM treatment produced highly significant gains in attachment level (0.88 mm vs. 0.57 mm; p=0.003) and pocket depth (0.87 mm vs. 0.63 mm; p=0.01) as compared to SRP alone. In summary, Periowave^TM demonstrated strong bactericidal activity against known periopathogens, and treatment of periodontitis using this system produced significantly better clinical outcomes than SRP alone. This, along with the absence of any adverse events in patients treated to date demonstrates that PDT is a safe and effective treatment for adult chronic periodontitis.

Photodynamic therapy (PDT) using topical aminolevulinic acid (ALA) is currently used as a clinical treatment for nonmelanoma skin cancers. In order to optimize PDT treatment, vascular shutdown early in treatment must be identified and prevented. This is especially important for topical ALA PDT where vascular shutdown is only temporary and is not a primary method of cell death. Shutdown in vasculature would limit the delivery of oxygen which is necessary for effective PDT treatment. Diffuse correlation spectroscopy (DCS) was used to monitor relative blood flow changes in Balb/C mice undergoing PDT at fluence rates of 10mW/cm² and 75mW/cm² for colon-26 tumors implanted intradermally. DCS is a preferable method to monitor the blood flow during PDT of lesions due to its ability to be used noninvasively throughout treatment, returning data from differing depths of tissue. Photobleaching of the photosensitizer was also monitored during treatment as an indirect manner of monitoring singlet oxygen production. In this paper, we show the conditions that cause vascular shutdown in our tumor model and its effects on the photobleaching rate.

The photophysical and photochemical properties of a newly developed photosensitizer α-(8-quinolinoxy) zinc phthalocyanine (α-(8-QLO)PcZn) were investigated for application in photodynamic therapy (PDT). The maximal Q band for α-(8-QLO)PcZn in dimethylformamide around 675 nm with the molar extinction coefficient of about 1.89×10⁵ mol^-1cm^-1. The fluorescence quantum and singlet oxygen (¹O₂) yields were determined to be 0.18±0.02 and 0.62±0.03, respectively. α-(8-QLO) PcZn has a diffuse cytoplasmic distribution in nasopharyngeal carcinoma C666-1 cells, and the efficient photodynamic cytotoxicity was observed. Our findings of this study suggest that α-(8-QLO)PcZn is a promising second-generation photosensitizer for PDT.

Doxorubicin (DOX), widely used in cancer chemotherapy, is limited by drug resistance and cardiac toxicity. Hyperthermia can aid the functionality of DOX, but current external heat delivery methods are hard to apply selectively and locally. Indocyanine green (ICG) absorbs near infrared light at 808nm (ideal for tissue penetration) and emits the energy as heat. These properties make it an ideal agent for rapid and localized hyperthermia. The purpose of this study was to investigate the in vitro cytotoxic effect of combined chemotherapy and hyperthermia to a DOX resistant ovarian cancer cell line (SKOV-3). The effects of laser-ICG photothermotherapy, which induces localized rapid heating, and an incubator, which induces a slow rate of heating, were compared. Cells were subjected to different concentrations of DOX and either 60 minutes in a 43°C incubator or to one minute at 43°C using 5μM of ICG and 808nm laser. SRB assay was used to measure cell growth. ICG itself without laser irradiation was not toxic to the cells. DOX by itself was cytotoxic with an IC₅₀ about 5μM. Both incubator and laser-ICG Hyperthermia in combination with DOX achieved significantly greater growth inhibition at all DOX concentrations compared to DOX alone. DOX combined with 60 minutes 43°C incubation lowered DOX IC₅₀ to about 1μM. The DOX IC₅₀ value with one minute laser-ICG was even lower (0.1μM) suggesting a synergistic effect between DOX and laser-ICG photothermotherapy. In conclusion, the combination of localized heating and chemotherapy may provide a valuable tool for cancer treatment with minimized toxic effect.

Materials have been developed which absorb radiation of one energy and emit light of another. We present a theoretical analysis of the use of these materials as light sources for photodynamic therapy (PDT). The advantage of this strategy is that radiation of higher particle energy (e.g. x ray or electron beam) or lower photon energy (e.g. infra-red) may have more favorable penetration in tissue or more readily available radiation sources than the radiation absorbed by the sensetizer. Our analysis is based on the transfer of energy from radiation fields to visible light. We analyze two scenarios: PDT pumped by (1) infrared light in a two-photon process and (2) ionizing radiation. In each case, we assume that the converting material and the sensitizer are matched sufficiently that the transfer of energy between them is essentially lossless. For the infinite and semiinfinite geometries typically used in PDT, we calculate the resulting photodynamic dose distribution, and compare it to the dose distribution expected for conventional PDT. We also calculate the dose of the incident beam (ionizing or infrared radiation) required to produce PDT-induced tumoricidal effects, and evaluate the expected toxicity in surrounding normal tissue. The toxicity is assumed to arise from thermal effects and acute ionizing radiation effects, for infrared and ionizing radiation, respectively. Our results predict that ionizing radiation will produce dose-limiting toxicity in most conventional geometries as a result of the high toxicity per unit energy of ionizing radiation. For infrared radiation, we predict that the toxicity can be moderated by proper choice of sensitizer and irradiation geometry and fractionation.

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and xray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns (with one exception), suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies with several human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone wings increases the photo-toxicity with little corresponding change in the dark cyto-toxicity.

We have proposed a new type of atrial fibrillation treatment with the early state photodynamic therapy (PDT), in which the interval time between the photosensitizer injection and irradiation is shorter than that in conventional way. We had demonstrated the acute electrical blockade by the PDT with talaporfin sodium and a red (670 nm) diode laser in ex vivo and in vivo experiment using rat normal myocardial tissue. The previous study of intracellular Ca2+ concentration measurement in rat cardiac myocytes during the PDT indicated that Ca2+ influx induced by the plasma membrane damage might be the main cause of the acute reaction of myocardial tissue. We found that the cell damage of cardiac myocytes triggered by the PDT was mainly influenced by the site where the photosensitizer exists. In this study, we examined the relationship between the sites of talaporfin sodium existing and cell death phenotypes in response to the PDT, in order to clarify the mechanism of the acute electrical blockade induced by the PDT in myocardial tissue. The talaporfin sodium fluorescence was observed after the various incubation times to visualize the time-lapse intracellular photosensitizer localization. The distribution of the photosensitizer was dependent on the incubation time. The change in intracellular Ca2+ concentration during the PDT was examined with a fluorescent Ca2+ indicator by a high-speed Nipkow confocal laser microscope (CSU-X1, Yokogawa Electric Company). We obtained the Ca2+ dynamics during the PDT which can explain the PDT-induced cell death pathways. We concluded that the Ca2+ influx induced by plasma membrane damage is the possible mechanism of the electrical blockade by the early state PDT.