This PDF file contains the front matter associated with SPIE Proceedings Volume 6845, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Photodynamic therapy (PDT) can target the members of the Bcl-2 family that protect cells from the initiation of apoptosis, a well-known death pathway. We examined the ability of HA14-1, a non-peptidic Bcl-2/Bcl-xL antagonist, to promote the efficacy of PDT. The photosensitizer was the porphycene CPO that causes photodamage to Bcl-2 located in the endoplasmic reticulum. Using low PDT doses together with LD5-20 concentrations of HA14-1, we found a marked synergistic effect. These results indicate that such an effect occurs when PDT is coupled with pharmacologic suppression of Bcl-2 function. HA14-1 is an unstable compound that decomposes in aqueous solution. This resulted in a rapid (~60 sec) burst of fluorescence that closely mimicked the properties of many fluorescent probes, but was traced to an effect produced when HA14-1 contacts serum proteins. Other Bcl-2 antagonists that do not produce any intrinsic fluorescence also promoted PDT efficacy. Moreover, briefly storing HA14-1 in aqueous medium until the fluorescent burst is over does not inhibit a subsequent synergistic promotion of PDT efficacy. We conclude that Bcl-2 antagonists can promote the efficacy of low-dose PDT in a manner unrelated to ROS production. The most likely explanation is an enhanced loss of anti-apoptotic Bcl-2 family function such that a threshold for initiation of apoptosis is crossed.

Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals Case Medical Center, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially the anti-apoptotic protein Bcl-2, are photodamaged. Apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered by the initial photodamage. A series of analogues of Pc 4 has been synthesized containing two axial ligands, one identical to the single ligand of Pc 4 and the other either the same as the Pc 4 ligand or bearing one or more hydroxyl groups. The hydroxyl-bearing axial ligands reduce the aggregation of the Pc in polar environments and direct the Pc's to lysosomes. Photoirradiation of cells that have taken up these Pc's into their lysosomes is 4-6 times more efficient at killing cells, as defined by loss of clonogenicity, than with Pc 4. Whereas PDT with Pc 4 photodamages Bcl-2 and Bcl-xL over the same dose response range as for cell killing, PDT with Pc 181 or the other lysosome-localizing Pc's causes much less photodamage to Bcl-2 relative to cell killing. Furthermore, in the case of the lysosome-bound Pc's, little or no caspase-3-dependent apoptosis is observed.

In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote mitochondrial permeabilization and apoptotic signaling. One important constituent seems to be Fe2+ that is taken up by mitochondria through the Ca2+ uniporter to promote mitochondrial ROS-dependent chain reactions. Lysosomal proteases may also directly promote apoptotic signaling, e.g., through cleavage/activation of the pro-apoptotic protein Bid.ÿÿÿ

Photodynamic Therapy (PDT) damages cells and tissues via generation of singlet oxygen (¹O₂). One target of ¹O₂ in biological systems may be proteins, and numerous studies have shown that ¹O₂ can inactivate enzymes. Whereas the reactions of ¹O₂ with His, Tyr, Met, Cys, and Trp are well known, the reaction with these same residues in proteins is less well studied. PDT induces apoptosis via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by activation of caspases. Because Cyt-c resides in the mitochondrial intermembrane space, associated with the inner membrane, it would be exposed to ¹O₂ generated by photoactivation of lipophilic photosensitizers like Pc 4 located in mitochondrial membranes. Studies by others have shown that ¹O₂ can oxidize the ferro-form of Cyt-c, inactivate the function of Cyt-c as an electron carrier, and result in the loss of His, Trp, Tyr, and Met residues. However, mass spectrometric evidence for modifications of specific amino acid side chains of proteins by ¹O₂ has not been reported to our knowledge. Here, we used MALDI-TOF-MS and LC-ESI-MS to study the reaction of ¹O₂ with amino acid residues within two model peptides in homogeneous medium and within Cyt-c in homogeneous media and in liposomes. The analyses revealed multiple oxidation products, including at least one His-derived product that is unique to singlet oxygen and not found following reaction with other reactive oxygen species. This product may serve as a marker of mitochondrial photodynamic damage.

An improved interstitial diffuse optical tomography (iDOT) system has been developed to characterize the optical properties of prostate gland during the photodynamic therapy (PDT). Multiple cylindrical light diffusers with different lengths (instead of point sources used in an earlier version) and isotropic detectors are introduced interstitially in the prostate gland in-vivo. During the data acquisition, linear sources and detectors are stepping into prostate sequentially controlled by a motorized system. A computerized multi-channel attenuator system is developed to automatically control the power strength of each linear source and on times to speed up data acquisition. Three dimensional optical properties are obtained by solving the inverse problem of steady-state diffusion equation based on an adjoint model with Moore- Penrose scheme. The convergence, accuracy and the speed of the algorithms are tested in mathematical phantoms and in prostate simulating phantoms with known optical properties. For comparison, the optical properties of tissue simulating phantoms are also reconstructed using iDOT with multiple isotropic point sources. Data acquisition time in iDOT using linear sources is at least 10 times faster than using the point sources with the total data acquisition time to be less than 1 minutes. Reconstruction results showed both algorithms can successfully recover the optical properties. Reconstruction using linear sources/detectors acquisition mode is 20 times faster than the point sources/detectors method (30 minutes vs. 4 hours on a 3.4 GHz Pentium PC with 4 GB memory). We have demonstrated that linear-source/detector acquisition mode out-performs the point-source mode, and is more practical to be implemented in the clinical settings.

Aminolevulinic Acid (ALA) is a prodrug which can be administered to cells, animals or patients after which it is transformed via the Heme synthesis pathway into the fluorescent molecule Protoporphyrin IX (PpIX). PpIX has been shown to be useful as both a photosensitizer for photodynamic therapy (PDT) and as a fluorescence imaging contrast agent. The ALA-PpIX system not only provides contrast for fluorescence imaging but also gives information about the metabolic activity of the imaged tissue and thus could be useful for monitoring cancer therapy. In the current study skin photobleaching was examined to determine if PpIX fluorescence contrast in malignant brain tumors could be better visualized noninvasively. Red light photobleaching decreased skin PpIX fluorescence and increased the ability to noninvasively quantify PpIX fluorescence in murine gliomas, as in vivo measurements of mean PpIX fluorescence more closely matched ex vivo quantification following skin photobleaching. Three doses of blue light photobleaching (4 J/cm2, 8 J/cm2 and 12 J/cm2) were tested and determined to give similar levels of skin photobleaching as well as a similar window of decreased skin PpIX fluorescence for noninvasive fluorescence imaging following the photobleaching dose administration.

Background: photodynamic therapy (PDT) mediated with vascular acting photosensitizer Tookad (pd-bacteriopheophorbide) was investigated as an alternative modality for treating prostate cancer. Our previous studies show that Tookad PDT can induce marked prostatic tissue lesion but minimal urethral lesion. In this study a transurethral procedure was used to evaluate the response of the prostatic urethra to direct urethral irradiation. Materials and Methods: Tookad solution (2.5 mg/ml) was administered (1 mg/kg) through IV catheter by an infusion pump over 10 min. A diffuser fiber (1 cm active length) was inserted into the prostatic urethra. The light irradiation (50 or 100 J/cm) started at 4 min after the onset of drug infusion. Urinalysis was performed for 24 - 48 h post PDT. One week after PDT, prostates (n = 4) were removed at necropsy and subjected to histopathological examination. Results: The cross section of prostates showed severe hemorrhagic and necrotic lesions on the right lobe. The diameter of the lesion, measured from urethra to capsule, was >13 mm for 50 J/cm treatment and >18 mm for 100 J/cm, respectively. Although underlying periurethral lesion was visible, the urethral surface was intact and prostatic urethra was open. Conclusions: The joint point of the diffuser tip and the guide fiber might be bent while passing through the sharp turn at the Ischial Arch, which could affect the light distribution and cause the asymmetric lesion. Nonetheless, the transurethral direct irradiation can induce marked prostatic tissue lesion but minimal urethral lesion.

Interstitial Photodynamic therapy (IPDT) has been under intense investigation in recent years, with multiple clinical trials underway. This effort has demanded the development of optimization strategies that determine the best locations and output powers for light sources (cylindrical or point diffusers) to achieve an optimal light delivery. Furthermore, we have recently introduced cylindrical diffusers with customizable emission profiles, placing additional requirements on the optimization algorithms, particularly in terms of the stability of the inverse problem. Here, we present a general class of linear feasibility algorithms and their properties. Moreover, we compare two particular instances of these algorithms, which are been used in the context of IPDT: the Cimmino algorithm and a weighted gradient descent (WGD) algorithm. The algorithms were compared in terms of their convergence properties, the cost function they minimize in the infeasible case, their ability to regularize the inverse problem, and the resulting optimal light dose distributions. Our results show that the WGD algorithm overall performs slightly better than the Cimmino algorithm and that it converges to a minimizer of a clinically relevant cost function in the infeasible case. Interestingly however, treatment plans resulting from either algorithms were very similar in terms of the resulting fluence maps and dose volume histograms, once the diffuser powers adjusted to achieve equal prostate coverage.

Endoscopic and interstitial diffuse optical tomography have been studied in clinical investigations for imaging prostate tissues, yet, there is no comprehensive comparison of how these two imaging geometries affect the quality of the reconstruction images. In this study, the effect of imaging geometry is investigated by comparing the cross-section of the Jacobian sensitivity matrix and reconstructed images for three-dimensional mathematical phantoms. Next, the effect of source-detector configurations and number of measurements in both geometries is evaluated using singular value analysis. The amount of information contained for each source-detector configuration and different number of measurements are compared. Further, the effect of different measurements strategies for 3D endoscopic and interstitial tomography is examined. The pros and cons of using the in-plane measurements and off-plane measurements are discussed. Results showed that the reconstruction in the interstitial geometry outperforms the endoscopic geometry when deeper anomalies are present. Eight sources 8 detectors and 6 sources 12 detectors are sufficient for 2D reconstruction with endoscopic and interstitial geometry respectively. For a 3D problem, the quantitative accuracy in the interstitial geometry is significantly improved using off-plane measurements but only slightly in the endoscopic geometry.

A light dosimetry system is developed for prostate PDT, which integrates four main components: a light fluence rate calculation engine, an optimization tool for treatment planning, a light delivery system, and an in vivo light fluence rate measurement system. Three-dimensional light fluence rate distribution in a prostate is calculated using a kernel algorithm, which takes into account of heterogeneous optical properties. A Cimmino optimization algorithm is used to optimize the parameters of the cylindrical diffusing fibers (CDFs) to generate uniform PDT dose (or light fluence rate under uniform drug distribution) to cover the heterogeneous prostate. The light delivery system is composed of a 12- channel beamsplitter and the intensities of each channel (i.e., source) are controlled individually by programmable motorized attenuators. Our tests show that the light fluence rate calculation is fast and the accuracy is close to that of a finite-element method model, and the approach that uses the treatment CDFs to determine optical properties, improves the accuracy of light fluence rate prediction. The light delivery system allows real time control of the light source intensities for both PDT dosimetry and PDT light delivery. Integrating the fast light fluence rate calculation, optimization, instant source intensity adjustment, and in vivo light fluence rate measurement, the dosimetry system is suitable for prostate PDT.

Over the past three years we have described the rationale for using new photosensitizers (PS) with greatly enhanced multi-photon absorption. In particular, we have synthesized new porphyrin-based photosensitizers that also incorporate small molecule targeting agents that direct the ensemble to over-expressed tumor receptor sites, as well as Near-infrared imaging agents that will allow practical image-guided two-photon PDT in the tissue transparency window (750-1000 nm) at laser fluences that are harmless to surrounding healthy tissue. We have previously shown (PW2006) successful treatment of human breast cancer models (MDA-MB-231) in SCID mice, and have recently extended these studies to the treatment of both human small cell (SC) (NCI-H69) and non-small cell (NSC) (A-459) models in SCID mice. We have demonstrated that lung cancer xenografts can be successfully treated by irradiating from the side of the mouse opposite the implanted tumor, thereby passing through ca. 2 cm of mouse skin, tissue and organs before encountering the bulk tumor. These results suggest that this technology can be used to treat deep subcutaneous spontaneous tumors in larger animal models (e.g. canine). We would also emphasize that the synthetic route to these triads attaches the targeting moiety in the last step of the synthesis, and can be easily changed, thus allowing a myriad of targeting agents to be employed, opening the door to the possibility of patient-specific PDT.

The development of multimodal molecular probes and photosensitizing agents for use in photodynamic therapy (PDT) is vital for optimizing and monitoring cytotoxic responses. We propose a combinatorial approach utilizing photosensitizing molecules that are both paramagnetic and luminescent with multimodal functionality to perturb, control, and monitor molecular-scale reaction pathways in PDT. To this end, a time-domain single photon counting lifetime apparatus with a 400 nm excitation source has been developed and integrated with a variable low field magnet (0- 350mT). The luminescence lifetime decay function was measured in the presence of a sweeping magnetic field for a custom designed photosensitizing molecule in which photoinduced electron transfer was studied The photosensitizer studied was a donor-acceptor complex synthesized using a porphyrin linked to a fullerene molecule. The magneto-optic properties were investigated for the free-base photosensitizer complex as well as those containing either diamagnetic (paired electron) or paramagnetic (unpaired electron) metal centers, Zn(II) and Cu(II). The magnetic field was employed to affect and modify the spin states of radical pairs of the photosensitizing agents via magnetically induced hyperfine and Zeeman effects. Since the Type 1 reaction pathway of an excited triplet state photosensitizer involves the production of radical species, lifetime measurements were conducted at low dissolved oxygen concentration (0.01ppm) to elucidate the dependence of the magnetic perturbation on the photosensitization mechanistic pathway. To optimize the magnetic response, a solvent study was performed examining the dependence of the emission properties on the magnetic field in solutions of varying dielectric constants. Lastly, the cytotoxicity in murine tumor cell suspensions was investigated for the novel porphyrin-fullerene complex by inducing photodynamic treatments and determining the associated cell survival.

A novel class of porphyrin-based near-infrared photodynamic therapy (PDT) sensitizers is studied. We achieve regressions of human small cell lung cancer (NCI-H69), non-small cell lung cancer (A 459) and breast cancer (MDAMB- 231) xenografts in SCID mice at significant tissue depth by irradiation with an amplified femtosecond pulsed laser at 800 nm wavelength. Significant tumor regressions were observed during the first 10-14 days post treatment. Tumor histopathology was consistent with known PDT effects, while no significant changes were noted in irradiated normal tissues. In vivo imaging studies using intravenous injections of fluorescent dextran demonstrated an early loss of tumor blood flow. RNA was isolated from NCI-H69 PDT treated SCID mouse xenografts and paired untreated xenografts at 4 hours post laser irradiation. Similarly RNA was isolated from PDT treated and untreated Lewis lung carcinomas growing in C57/Bl6 mice. Expression profiling was carried out using Affymetrix^TM human and mouse GeneChips®. Cluster analysis of microarray expression profiling results demonstrated reproducible increases in transcripts associated with apoptosis, stress, oxygen transport and gene regulation in the PDT treated NCI-H69 samples. In addition, PDT treated Lewis lung carcinomas showed reproducible increases in transcripts associated with immune response and lipid biosynthesis. PDT treated C57/Bl6 mice developed cytotoxic T cell activity towards this tumor, while untreated tumor bearing mice failed to do so.

Photodynamic Therapy (PDT) is an emerging and promising therapeutic modality for treatment of a wide variety of malignant and nononcologic tumors, as well as in the treatment of infected skin ulcers. This study evaluated the effectiveness of the PDT to treat a chronic skin wound that had been already subjected to several clinical and surgical type treatments in a dog. The animal with an infected chronic skin wound with 8 cm diameter in the left leg received an injection of an aqueous solution of 1% methylene blue (MB) with 2% lidocaine into the lesion. After MB injection the wound was irradiated using a LED (LED-VET MMOptics(r)) with a wavelength between 600 and 700 nm, 2 cm diameter circular light beam, of 150 mW of power, light dose of 50 J/cm². After 3 and 6 weeks PDT was repeated and the wound was re-evaluated. Complete healing was achieved 10 weeks after the first procedure.

Introduction. Life induced fluorescence (LIFE) diagnostics can be used as an imaging system of precancerous and neoplastic lesions of the oral mucosa. Neoplastic lesions are visible in pseudo colours, healthy tissue in green colour and abnormal tissue in red colour. All the observed colours present different intensity. Colour intensity is relevant to the grade of dysplasia, carcinoma progress and is called Numerological Value of Color Index (NCV). The aim of our study was to find correlation between autofluorescence diagnostics combined with NCV assessment and histopathological findings of taken specimen biopsies. Patients and methods. 10 patients participated in our study. Lesions affected a variety of intraoral sites. The most common location was: buccal, gingival and mandibular mucosa. Patients were examined using Life Induced Fluorescence diagnosis (400 - 750 nm wavelength) with Numerological Value of Color index (NCV) using Onco LIFE system. Afterwards the specimen biopsies from the lesions were taken and histopathological examination was performed. Results. Different NCV and dependence of NCV on the histopathological findings were observed. Conclusion. Diagnostic procedures with the application of white-light imaging with LIFE imaging is not only a significantly faster method and a better diagnostic tool of preneoplastic and neoplastic lesions, but there exist also correlations between measured NCV and histopathological diagnosis. The farther investigations are necessary in order to prove these preliminary findings.

Tumor oxygen depletion plays an important role in the process of Photodynamic Therapy (PDT). The paper focuses on the improvement of the lighting mode to carry out this cancer therapy more effectively in low oxygen content. The effect of interstitial lighting was compared with that of continuous lighting in different oxygen density measured with a homemade device in PDT. 90 mice were divided into 3 groups: the contrast group, the continuous lighting group and the interstitial lighting group. The initial oxygen content was measured with a homemade device before the treatment. To examine the different effects, both the interstitial lighting and the continuous lighting have the same fluent rates (30mW/cm2, 32.4J/ cm2). The continuous lighting lasted 18 minutes while the interstitial lighting lasted 36 minutes with 1 second's idle time and 1 second's effective time of each pulse. The result shows that the volume of tumor doubling duration in interstitial lighting group is longer in the condition of low initial oxygen content. Thus with low initial oxygen content, the interstitial lighting is more effective than the continuous lighting during PDT.

The Optical Pharmacokinetic System (OPS) is an elastic-scattering spectroscopic device that is capable of detecting changes in hemoglobin saturation in tissue in vivo. This measurement is important to the field of photodynamic therapy (PDT) because it may be possible to use knowledge of tissue oxygen concentration to improve treatment effect. The OPS measures a 'bulk' signal that is representative of the total tissue volume sampled optically. This measurement may not be sensitive to the presence or development of small, non-uniform hypoxic regions within tissue, and therefore, the clinical relevance of such a measurement is not well understood. This study utilizes mathematical models to investigate the sensitivity of the OPS to chronic hypoxic regions that exist in tumor tissue at steady state and acute hypoxic regions caused by PDT-induced damage. A Monte Carlo model of light propagation is used to emulate the measurement of tissue by the OPS. Tissue geometry is constructed to mimic the tumor microvascular environment, with discrete blood vessels interspersed throughout. A finite element-based transport model is used to describe spatial distributions of oxygen, reactive oxygen species, and hemoglobin saturation throughout the tissue at steady state and following the PDT reaction. PDT-induced damage is estimated and used to approximate the effect of vascular damage on tissue oxygen concentration, thereby simulating acute hypoxia. The volume-averaged hemoglobin saturation measured by the OPS shows potential to identify the presence hypoxic vessels in the chronic case. However, results suggest that the clinical utility of the OPS to detect PDT-induced hypoxia may be limited.

Photodynamic therapy dosimetery and treatment planning is affected by the concentration of photosensitizer in a given tissue, and these values are often estimated based on measurements of fluorescence in the region to be treated. Some studies with benzoporphyrin derivate monoacid ring a (BPD-MA) showed a significant increase in fluorescence quantum yield with deoxygenation of the solution, indicating a possible oxygen sensitive switch in intersystem crossing or reverse intersystem crossing. The experiments done in this paper show that at oxygenation levels found in vivo the variation in fluorescence quantum yield of liposomal BPD-MA (verteporfin) is negligible for changes in solution oxygenation. The results from all of the experiments show that it is not necessary to measure the oxygenation of tissues when calculating the concentration of verteporfin from fluorescence measurements, so that dosimetry calculations based upon photosensitizer levels would not be affected by the tissue oxygenation. This greatly simplifies the dosimetry process with verteporfin.

Interstitial photodynamic therapy (iPDT) is a promising minimally invasive treatment modality for locally confined prostate cancer. Therapeutically excited at 635nm, the photophysical properties of SL-052 (a novel hypocrellin derivative photosensitizer) lend themselves uniquely to iPDT, facilitating real-time monitoring. Under 635nm excitation, SL-052 exhibits near infrared fluorescence, allowing both photosensitizer fluorescence and tissue transmissivity to be continuously monitored. The absorption and fluorescence characteristics of SL-052 in vivo and in vitro are first illustrated. SL-052 mediated iPDT of canine prostate was performed with a novel switched light delivery system and novel intra-arterial drug delivery method. A preliminary examination of the dosimetric properties of intra-arterial iPDT is presented, focusing on transmissivity dynamics. Spectrofluorimetry results relating specifically to the unique photophysical properties of SL-052 iPDT are also included.