This PDF file contains the front matter associated with SPIE Proceedings Volume 8931, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

Photokilling depends on the generation of death signals after photosensitized cells are irradiated. A variety of intracellular organelles can be targeted for photodamage, often with a high degree of specificity. We have discovered that a low level of photodamage directed against lysosomes can sensitize both a murine hepatoma cell line (in 2D culture) and an inflammatory breast cancer line of human origin (in a 3D model) to subsequent photodamage directed at mitochondria. Additional studies were carried out with hepatoma cells to explore possible mechanisms. The phototoxic effect of the ‘sequential targeting’ approach was associated with an increased apoptotic response. The low level of lysosomal photodamage did not lead to any detectable migration of Fe⁺⁺ from lysosomes to mitochondria or increased reactive oxygen species (ROS) formation after subsequent mitochondrial photodamage. Instead, there appears to be a signal generated that can amplify the pro-apoptotic effect of subsequent mitochondrial photodamage.

Photodynamic therapy (PDT) is an emerging medical treatment that uses photosensitizers (drug) which are activated by laser light for the generation of cytotoxic free radicals and singlet oxygen molecules that cause tumor cell death. In the recent years, there has been a focus on using and improving an industrial colorant termed phthalocyanines as a prospective photosensitizer because of its unique properties. This in vitro study investigated the photodynamic effect of indium (InPcCl) and iron (FePcCl) phthalocyanine chlorides on human skin cancer cells (melanoma). Experimentally, 2 x 104 cells/ml were seeded in 24-well tissue culture plates and allowed to attach overnight, after which cells were treated with different concentrations (2 μg/ml - 100 μg/ml) of InPcCl and FePcCl. After 2 h, cells were irradiated with constant light doses of 2.5 J/cm², 4.5 J/cm² and 8.5 J/cm2 delivered from a diode laser. Post-irradiated cells were incubated for 24 h before cell viability was measured using the MTT Assay. At 24 h after PDT, irradiation with a light dose of 2.5 J/cm² for each photosensitizing concentration of InPcCl and FePcCl produced a significant decrease in cell viability, but when the treatment light dose was further increased to 4.5 J/cm² and 8.5 J/cm² the cell survival was less than 55% for photosensitizing concentrations of InPcCl and FePcCl from 4 μg/ml to 100 μg/ml. This PDT study concludes that low concentrations on InPcCl and FePcCl activated with low level light doses can be used for the effective in vitro killing of melanoma cancer cells.

We used three dimensional cell cultures (3D) based on the magnetic levitation method (MLM) to evaluate cytotoxicity of photodynamic therapy (PDT). First, we decorated Hep G2 and MDA-MB-321 cells with NanoShuttle by introducing it in the media and incubated overnight. Next day, we transferred the cells to a 6-well plate and placed a magnetic driver on the top of the plate to start levitation. We monitored the formation of the 3D cell culture by optical microscopy and after four days, we added the photosensitizer Photogem (PG) in the culture media in concentrations of 50, 25, 12.5, 6.25μg/ml. We incubated them for 24 hours, after that we washed the cultures with PBS and added fresh media. Samples were then illuminated for 600s using a 630nm LED-based device, generating light intensities of 30 mW/cm² in a total light fluence of 18 J/cm². Following the illumination, we added fresh media, and 30 hours later, the 3D structures were broken using a pipettor and the cells seeded in 96 well plates, 10⁵ cells per well, with a magnetic drive placed on the bottom of the plate to create cell culture dots. After 24 hours, we used a MTT assay to evaluate PDT cytotoxicity. The PDT effect, evaluated by the half maximal effective concentration (EC₅₀), in MDA-MB-231 cells (EC₅₀ =3.14 μg/ml) is more aggressive compared to the effect of PDT in Hep G2 cells (EC₅₀ = 7.48 μg/ml). It suggests that the cell culture structure and its interaction facilitated the PG uptake and consequently elevated the Photodynamic effect for MDA-MB-231.

Photodynamic Therapy (PDT) is a type of cancer treatment that is based on the interaction of light (with specific wavelength), a photosensitizing agent and molecular oxygen. The photosensitizer (PS) is activated by light and reacts with oxygen resulting in the production of singlet oxygen that is highly reactive and responsible for the cell death. The Chick Chorioallantoic Membrane (CAM) model is a transparent membrane that allows visualization and evaluation of blood vessels and structural changes, where a tumor model was developed. Two induction tumor models were investigated: tumor biopsy or cell culture. It was used a murine melanoma cell B16F10 in culture and a biopsy from a xenograft tumor in hairless mouse. Two PS were tested: Photodithazine® and Photogem®, a chlorine and porphyrin compounds, respectively. Using intravenous administration, the light-drug interval was of 30 minutes, 1 and 3 hours. Illumination was performed at 630 nm and 660 nm, and the vascular and tumor response was monitored and analyzed. The PS distribution was checked with confocal microscopy. This model can be useful to study several parameters of PDT and the effect of this therapy in the cancer treatment since it allows direct visualization of its effects.

There has been significant effort over the past two decades in the treatment of malignancies of epithelial origin, including some of the most devastating of cancers, such as colorectal cancer (CRC), squamous call carcinoma of the head and neck (HNSCC), and carcinomas of the pancreas, lungs, (both Small Cell and Non-Small Cell), renal cell, prostate, bladder and breast. Recurring, refractory HNSCC is a particularly difficult cancer to treat once the tumors recur due to mutations that are resistant to repeat chemotherapy and radiation. In addition, repeat surgery is often difficult due to the requirement of significant surgical margins that may not be possible due to the attending potential functional deficits (e.g., salivary glands, nerves and major blood vessels in confined areas). In this study FaDu HNSCC xenograft tumors in SCID mice were imaged, and “optical”, as opposed to “surgical” margins defined for the tumor being treated. The subsequent two-photon treatment irradiation was computer-controlled to carry out the tumor treatment by rastering the laser beam throughout the tumor volume plus the defined optical margins simultaneously. In our initial studies, up to 85% regression in tumor volume was observed in 5 days post PDT, with complete tumor regression in 18 days. No re-growth was observed up to 41 days post-PDT, with little or no scarring and complete hair re-growth. However, competition between imaging and PDT moieties was also observed in some mouse models, possibly favoring tumor re-growth. Strategies to selectively optimize the PDT effect will be discussed.

Oxygen plays a major role in cancer biology and tumor progression. In PDT, the reduction in efficacy is directly related to lack of oxygen because its molecular mechanism relies on oxygen as an energy mediator. Measuring tumor oxygenation can provide physicians with better diagnosis and optimization of treatment plans. However, clinical tools for directly assessing tissue oxygenation are limited. The gold standard is oxygen needle electrode, which is invasive and measures oxygen level at a single location. We present our work on developing a combined treatment-imaging modality that integrates PDT and photoacoustic oxygen imaging. We propose a system designed for clinical treatments of cancer of the oral cavity. Tissue oxygen imaging is performed by applying Photoacoustic Lifetime Imaging (PALI). This technology relies on photoacoustic probing of oxygen-dependent excitation lifetime of Methylene Blue. The dye is excited by the same wavelength of illumination source for PDT. Once excited, the population of photosensitizer molecules at triplet state has a lifetime depending on the oxygen level. The transition from excited triplet state to ground state can be probe by another laser, which generate photoacoustic signal that is used to map the lifetime. The lifetime map is then converted to pO2 distribution. We expect that PDT efficacy can be improved by applying PALI imaging feedback in real-time to determine, and individually optimize, O2-enriched gas breathing parameters and PDT light-dose during treatment. Successful implementation of PALI in PDT can also drive its application in guiding other cancer treatments that are affected by hypoxia.

Macroscopic modeling of singlet oxygen (¹O₂) is of particular interest because it is the major cytotoxic agent causing biological effects for type II photosensitizers during PDT. We have developed a macroscopic model to calculate reacted singlet oxygen concentration ([1O2]_rx for PDT. An in-vivo RIF tumor mouse model is used to correlate the necrosis depth to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include 4 photosensitizer specific photochemical parameters along with the apparent singlet oxygen threshold concentration. Photosensitizer specific model parameters are determined for several type II photosensitizers (Photofrin, BPD, and HPPH). The singlet oxygen threshold concentration is approximately 0.41 - 0.56 mM for all three photosensitizers studied, assuming that the fraction of singlet oxygen generated that interacts with the cell is (f = 1). In comparison, value derived from other in-vivo mice studies is 0.4 mM for mTHPC. However, the singlet oxygen threshold doses were reported to be 7.9 and 12.1 mM for a multicell in-vitro EMT6/Ro spheroid model for mTHPC and Photofrin PDT, respectively. The sensitivity of threshold singlet oxygen dose for our experiment is examined. The possible influence of vascular vs. apoptotic cell killing mechanism on the singlet oxygen threshold dose is discussed using the BPD with different drug-light intervals 3 hrs vs. 15 min. The observed discrepancies between different experiments warrant further investigation to explain the cause of the difference.

Knowledge of optical properties is required to determine light dose in photodynamic therapy. We have designed an interstitial optical probe, consisting of six helically arranged side-firing fibers enclosed in a 1.1 mm diameter encapsulant, that can be used to determine these values. White light is delivered by one fiber and detected by the others. Based on a Monte Carlo (MC) model of the probe, the absorption (μa) and reduced scattering (μs') coefficients of the sample are determined. Recovery was verified in tissue-simulating phantoms containing MnTPPS or intact human erythrocytes as absorbers and Intralipid as scatterer. Mean errors in recovery of μa and μs' were 9% and 19%, respectively. In phantoms containing erythrocytes, hemoglobin oxygen saturation was recovered with mean error of 12%. Using the MC model, we mapped the volumes sampled by particular spectroscopy fibers. For μa = 0.1 cm^-1 and μs' = 20cm^-1, 49% of photon packets detected at the fiber adjacent to the source sampled a radius further than 5 mm from the probe, while 24% of photon packets sampled further than 7.5 mm. When μs' was reduced to 10 cm^-1, 54% of photon packets traversed a radius greater than 5 mm from the probe and 29% sampled further than 7.5 mm. Changing the value of μa to 0.2 cm^-1 did not have an effect on the sampled volume. We also provide a new probe design that aims to improve upon the accuracy of the current probe by incorporating a wider range of source-detector separations.

Background and aims: Nanoparticles have been explored recently as an efficient delivery system for photosensitizers in photodynamic therapy. In this study, polyhematoporphyrin (C₃₄H₃₈N₄NaO₅,) was loaded into hollow silica nanoparticles (HSNP) by one-step wet chemical-based synthetic route. We evaluate the efficacy and safety of polyhematoporphyrin-loaded HSNP with hepatobiliary malignant cells and in vivo models. Methods: Human liver cancer, cholangiocarcinoma and gallbladder cancer cells were cultured with the HSNP and cellular viability was determined by MTT assay. Apoptotic and necrotic cells were measured by flow cytometry. Finally, we investigate its effect in vivo. Results: In MTT assay, the cell viability of QBC939, Huh-7, GBC-SD and HepG2 cells of the HSNP was 6.4±1.3%, 6.5±1.2%, 3.7±1.2% and 4.7±2.0%, respectively, which were significant different from that of free polyhematoporphyrin 62.4±4.7%, 62.5±6.0%, 33.4±6.5% and 44.3±1.9%. Flow cytometry demonstrated the laser-induced cell death with polyhematoporphyrin-loaded HSNP was much more severe. Similarly, in vivo results of each kind of cell revealed 14 days post-photoradiated, tumor sizes of the HSNP group were significantly smaller. Administration of the HSNP without illumination cannot cause killing effect both in vitro and in vivo experiments. Conclusions: HSNP is a desirable delivery system in photodynamic therapy for hepatobiliary malignacies, with improved aqueous solubility, stability and transport efficiency of photosensitizers.

The increasing prevalence of antibiotic resistance requires new approaches also for the treatment of infectious keratitis. Photodynamic Inactivation (PDI) using the photosensitizer (PS) Chlorin e6 (Ce6) was investigated as an alternative to antibiotic treatment. An in-vitro cornea model was established using porcine eyes. The uptake of Ce6 by bacteria and the diffusion of the PS in the individual layers of corneal tissue were investigated by fluorescence. After removal of the cornea’s epithelium Ce6-concentrations < 1 mM were sufficient to reach a penetration depth of 500 μm. Liquid cultures of microorganisms were irradiated using a specially constructed illumination chamber made of Spectralon(R) (reflectance: 99 %), which was equipped with high power light emitting diodes (λ = 670 nm). Clinical isolates of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) from keratitis patients were tested in liquid culture against different concentrations of Ce6 (1 - 512 μM) using 10 minutes irradiation (E = 18 J/cm² ). This demonstrated that a complete inactivation of the pathogen strains were feasible whereby SA was slightly more susceptible than PA. 3909 mutants of the Keio collection of Escherichia coli (E.coli) were screened for potential resistance factors. The sensitive mutants can be grouped into three categories: transport mutants, mutants in lipopolysaccharide synthesis and mutants in the bacterial SOS-response. In conclusion PDI is seen as a promising therapy concept for infectious keratitis.

Photodynamic therapy using aminoluvelinic acid (ALA) is an FDA-approved treatment for actinic keratoses, pre-cancerous skin lesions which pose a significant risk for immunocompromised individuals, such as organ transplant recipients. While PDT is generally effective, response rates vary, largely due to variations in the accumulation of the photosensitizer protoporphyrin IX (PpIX) after ALA application. The ability to quantify PpIX production before treatment could facilitate the use of additional interventions to improve outcomes. While many groups have demonstrated the ability to image PpIX in the clinic, these systems generally require darkening the room lights during imaging, which is unpopular with clinicians. We have developed a novel wide-field imaging system based on pulsed excitation and gated acquisition to image photosensitizer activity in the skin. The tissue is illuminated using four pulsed LED’s to excite PpIX, and the remitted light acquired with a synchronized ICCD. This approach facilitates real-time background subtraction of ambient light, precluding the need to darken the exam room. Delivering light in short bursts also allows the use of elevated excitation intensity while remaining under the maximum permissible exposure limits, making the modality more sensitive to photosensitizer fluorescence than standard approaches. Images of tissue phantoms indicate system sensitivity down to 250nM PpIX and images of animals demonstrate detection of PpIX fluorescence in vivo under normal room light conditions.

Photodynamic therapy (PDT) is a technique that has been used for the treatment of tumors, especially in Gynecology. The photodynamic reaction is based on the production of reactive oxygen species after the activation of a photosensitizer. Advantages of the PDT in comparison to the surgical resection are: ambulatory treatment and tissue recovery highly satisfactory, through a non-invasive procedure. The cervical intraepithelial neoplasia (CIN) grades I and II presents potential indications for PDT. The aim of the proposed study is to evaluate the safety and efficacy of the PDT for the diagnostics and treatment of CIN I and II. The equipment and the photosensitizer are produced in Brazil with a representative low cost. It is possible to visualize the fluorescence of the cervix and to treat the lesions, without side effects. The proposed clinical protocol shows great potential to become a public health technique.

Patients with non-small cell lung cancer (NSCLC) with pleural dissemination have very limited survivals often of just 6-9 months. Prior reports of aggressive surgical resection of pleural metastases have shown no consistent improvements in overall survival and very high rates of local recurrences. Based on this and the generally very diffuse pleural dissemination seen in patients, chemotherapy and palliative interventions are standard of care. By attempting to sterile microscopic residual disease after surgical resection, intraoperative photodynamic therapy (PDT) could improve local pleural control and overall survival compared with surgery alone for patients with NSCLC with pleural metastasis. Prior attempts to demonstrate an improvement in clinical outcomes with PDT as an intraoperative adjuvant combined with definitive surgery to treat pleural malignancies have not been successful, perhaps due, in part, to limited ability to perform real-time dosimetry and ensure adequate and even light distribution throughout the chest cavity. A stratified phase II trial assessed the efficacy of definitive surgery and intraoperative PDT with real-time dosimetry in patients with NSCLC with pleural dissemination demonstrated prolonged local control and a higher than expected 21.7-month median survival from the time of surgery and PDT among 22 enrolled patients. This is the first ever report describing optimal methods, techniques, and dosimetry that could be used to safely and reproducibly deliver intraoperative PDT to the chest cavity as part of multimodality therapy for NSCLC with pleural metastasis.

Photodynamic therapy (PDT) has emerged as an alternative approach to chemotherapy and radiotherapy for cancer treatment. The photosensitizer (PS) is perhaps the most critical component of PDT, and continues to be an area of intense scientific research. Traditionally, PS molecules (e.g. porphyrins) have dominated the field. Nevertheless, these PS agents have several disadvantages, with low water solubility, poor light absorption and reduced selectivity for targeted tissues being some of the main drawbacks. Polysilsesquioxane (PSilQ) nanoparticles are crosslinked homopolymers formed by the condensation of functionalized trialkoxysilanes or bis(trialkoxysilanes). We believe that PSilQ particles provide an interesting platform for developing PS nanocarriers. Several advantages can be foreseen by using this platform such as carrying a large payload of PS molecules; their surface and composition can be tailored to develop multifunctional systems (e.g. target-specific); and due to their small size, nanoparticles can penetrate deep into tissues and be readily internalized by cells. In this work, PSilQ nanoparticles with a high payload of photosensitizers were synthesized, characterized, and applied in vitro. The network of this nanomaterial is formed by protoporphyrin IX (PpIX) molecules chemically connected via a redox-responsive linker. Under reducing environment such as the one found in cancer cells the nanoparticles can be degraded to efficiently release single photosensitizers in the cytoplasm. The phototoxicity of this porphyrin-based PSilQ nanomaterial was successfully demonstrated in vitro using human cervical (HeLa) cancer cells. We envision that this platform can be further functionalized with polyethylene glycol (PEG) and targeting ligands to improve its biocompatibility and target specificity.

In the operating room, time is extremely precious, and the speed of one’s data acquisition system often determines whether the data will be taken or not. Our multichannel robotic platform addresses this issue by optimizing source and detector scanning procedures. Up to 16 fibers can be moved independently with resolution of 0.05 mm and speed of 50 mm/s using motors with position feedback. The initial fiber alignment employs a light beam/optical detector system for identical positioning of all motors. Peak and edge detection algorithms, for point and linear sources, are used with multiple fibers simultaneously for fast realignment of sources and detectors. The robotic platform is used to perform Diffuse Optical Tomography (DOT) measurements in solid prostate phantoms with both homogenous and inhomogeneous Optical Properties (OP). Correct positioning is critical for the accurate recovery of the OP. The light fluence rate distribution is determined by scanning multiple detector fibers simultaneously along lit linear sources placed throughout the phantom volume inside catheter needles. The scanning time for the entire DOT is about 10 seconds after the initial alignment. The OP distribution reconstruction is based on the steady-state light diffusion equation. The inverse interstitial DOT problem is solved using NIRFAST. The optical properties are recovered by iterative minimization of the difference between measured and calculated light fluence rates. Recovered OP agree with the actual values within 10%. The OP corrections are used to significantly improve light fluence accuracy for the entire volume of bulk tumor.

We have reported a new molecular-targeted cancer phototherapy, photoimmunotherapy (PIT), which killed implanted tumors in mice without side-effects. To understand the mechanism of cell killing with PIT, three-dimentional dynamic low-coherence quantitative phase microscopy (3D LC-QPM), a device developed by Hamamatsu Photonics K.K, was used to detect morphologic changes in cancer cells during PIT. 3T3/HER2 cells were incubated with anti-HER2 trastuzumab-IR700 (10 μg/mL, 0.1 μM as IR700) for 24 hours, then, three-dimensionally imaged with the LC-QPM during the exposure of two different optically filtered lights for excitation of IR700 (500-780 nm) and imaging (780-950 nm). For comparison with traditional PDT, the same experiments were performed with Photofrin (10 and 1 μM). Serial changes in the cell membrane were readily visualized on 3D LC-QPM. 3T3/HER2 cells began to swell rapidly after exposure to 500-780 nm light excitation. The cell volume reached a maximum within 1 min after continuous exposure, and then the cells appeared to burst. This finding suggests that PIT damages the cell membrane by photo-reaction inducing an influx of water into the cell causing swelling and bursting of the cells. Interestingly, even after only 5 seconds of light exposure, the cells demonstrated swelling and bursting albeit more slowly, implying that sufficient cumulative damage occurs on the cell membrane to induce lethal damage to cells even at minimal light exposure. Similar but non-selective membrane damage was shown in PDT-treated cells Photofrin. Thus, PIT induces sufficient damage to the cell membrane within 5 seconds to induce rapid necrotic cell death which can be observed directly with 3D LC-QPM. Further investigation is needed to evaluate the biochemical mechanisms underlying PIT-induced cellular membrane damage.

Treatment monitoring of Aminolevunilic-acid (ALA) - Photodynamic Therapy (PDT) of basal-cell carcinoma (BCC) calls for superficial and subsurface imaging techniques. While superficial imagers exist for this purpose, their ability to assess PpIX levels in thick lesions is poor; additionally few treatment centers have the capability to measure ALA-induced PpIX production. An area of active research is to improve treatments to deeper and nodular BCCs, because treatment is least effective in these. The goal of this work was to understand the logistics and technical capabilities to quantify PpIX at depths over 1mm, using a novel hybrid ultrasound-guided, fiber-based fluorescence molecular spectroscopictomography system. This system utilizes a 633nm excitation laser and detection using filtered spectrometers. Source and detection fibers are collinear so that their imaging plane matches that of ultrasound transducer. Validation with phantoms and tumor-simulating fluorescent inclusions in mice showed sensitivity to fluorophore concentrations as low as 0.025μg/ml at 4mm depth from surface, as presented in previous years. Image-guided quantification of ALA-induced PpIX production was completed in subcutaneous xenograft epidermoid cancer tumor model A431 in nude mice. A total of 32 animals were imaged in-vivo, using several time points, including pre-ALA, 4-hours post-ALA, and 24-hours post-ALA administration. On average, PpIX production in tumors increased by over 10-fold, 4-hours post-ALA. Statistical analysis of PpIX fluorescence showed significant difference among all groups; p<0.05. Results were validated by exvivo imaging of resected tumors. Details of imaging, analysis and results will be presented to illustrate variability and the potential for imaging these values at depth.

Helicobacter pylori (HP), a gram-negative microaerophilic bacterium located in gastric mucosa, plays an im- portant role in gastro carcinogenesis. Due to the increasing emergence of antibiotic resistance, photodynamic inactivation of bacteria presents a new approach to treat bacterial infections, like HP. In vitro experiments were performed to determine the irradiation conditions for a complete inactivation of HP with the photosensitizer Chlorin e6 (Ce6). The HP strain CCUG 38770 (Culture Collection, University of Gothenburg, Sweden) was routinely cultured under microaerophilic conditions, suspended in sodium chloride, incubated with Ce6 and irradiated briefly with red light of the appropriate wavelength of λ = 660 nm. Series of measurements of different Ce6-concentrations (0.1 μM - 100 μM) were carried out, whereby the incubation time was kept constant at 1 min. The absorbed energy dose has been selected in varying the irradiation time (1 s - 300 s) and the power density (4.5 mW/cm² - 31 mW/cm² ). Quantification of inactivation was performed by enumeration of the grown colonies. In addition, the accumulation of Ce6 in HP cells was studied more precisely by uorescence spectroscopy. With a Ce6 concentration of 100 μM and a power density of 9 mW cm² , a 6-log10 reduction in the survival rate of HP was achieved within 30 seconds of irradiation. In conclusion the most relevant factor for the inactivation of HP is the exposure time of irradiation, followed by the concentration of Ce6 and the light intensity. Further studies with HP strains obtained from patient specimens are under current investigation.

An infrared (IR) camera system has been developed for use in pleural photodynamic therapy (PDT). This system was introduced to pleural PDT to provide uniform light dose distribution to ensure predictable PDT outcome. Light is delivered through a fiber that is in an endotracheal (ET) tube filled with Intralipid as scattering media. A tracking tool is attached to the ET tube to monitor the position of the optical fiber based point source. An anisotropic light distribution model is introduced to correct the angle dependent light distribution due to a capped end by design of the ET tube, which scatters light differently than the sides. In this study, the anisotropic nature of the balloon was characterized and incorporated into the calculation for light fluence during treatment. This model is verified by the light dose calculation from a phantom study. Furthermore, a new tracking tool was designed with multiple faces to increase the angular field of view and thus collect more viable data during treatment. The new tracking tool is directly entered into the ET tube with the light delivering fiber, thus eliminating the need to calibrate the laser source position prior to treatment via an optimization method. With this improved system, the calculated light fluence and the measured isotropic detector readings are more accurately matched.

Singlet oxygen (¹O₂) is the major cytotoxic species producing PDT effects, but it is difficult to monitor in vivo due to its short life time in real biological environments. Mathematical models are then useful to calculate ¹O₂ concentrations for PDT dosimetry. Our previously introduced macroscopic model has four PDT parameters: ξ, σ, β and g describing initial oxygen consumption rate, ratio of photobleaching to reaction between ¹O₂ and cellular targets, ratio of triplet state (T) phosphorescence to reaction between T and oxygen (³O₂), and oxygen supply rate to tissue, respectively. In addition, the model calculates a fifth parameter, threshold ¹O₂ dose ([¹O₂]rx,sd). These PDT parameters have been investigated for HPPH using radiation-induced fibrosarcoma (RIF) tumors in an in-vivo C3H mouse model. In recent studies, we additionally investigated these parameters in human non-small cell lung carcinoma (H460) tumor xenografts, also using HPPH-mediated PDT. In-vivo studies are performed with nude female mice with H460 tumors grown intradermally on their right shoulders. HPPH (0.25 mg/kg) is injected i.v. at 24 hours prior to light delivery. Initial in vivo HPPH concentration is quantified via interstitial HPPH fluorescence measurements after correction for tissue optical properties. Light is delivered by a linear source at various light doses (12-50 J/cm) with powers ranging from 12 to 150 mW per cm length. The necrosis radius is quantified using ScanScope after tumor sectioning and hematoxylin and eosin (H and E) staining. The macroscopic optimization model is used to fit the results and generate four PDT parameters. Initial results of the parameters for H460 tumors will be reported and compared with those for the RIF tumor.

The cell killing mechanism of benzoporphyrin derivative monoacid ring A (BPD) is known to be predominantly apoptotic or vascular, depending on the drug-light interval (DLI). With a 3 hour DLI, necrosis develops secondary to tumor cell damage, while with a 15 minute DLI, necrosis results from treatment-created vascular damage. The purpose of this study is to examine if the different mechanisms of cell death will affect the photochemical parameters for the macroscopic singlet oxygen model. Using the RIF model of murine fibrosarcoma, we determined the four photochemical parameters (see manuscript) and the threshold singlet oxygen dose for BPD-mediated PDT through evaluation of the extent of tumor necrosis as a function of PDT fluence rate and total fluence. Mice were treated with a linear source at fluence rates from 12-150 mW/cm and total fluences from 24-135 J/cm. BPD was administered at 1mg/kg with a 15 minute DLI, followed by light delivery at 690nm. Tumors were excised at 24 hours after PDT and necrosis was analyzed via HE staining. The in-vivo BPD drug concentration is determined to be in the range of 0.05-0.30 μM. The determination of these parameters specific for BPD and the 15 minute DLI provides necessary data for predicting treatment outcome in clinical BPD-mediated PDT. Photochemical parameters will be compared between 1mg/kg DLI 3 hours and 1mg/kg DLI 15 minutes.