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Significance: Photodynamic therapy (PDT) can be targeted toward different subcellular localizations, and it is proposed that different subcellular targets vary in their sensitivity to photobiological damage. Since singlet oxygen ( 1 O 2 ) has a very short lifetime with a limited diffusion length in cellular environments, measurement of cumulative 1 O 2 luminescence is the most direct approach to compare the PDT sensitivity of mitochondria and plasma membrane., Approach: PDT-generated near-infrared 1 O 2 luminescence at 1270 nm was measured together with cell viability for 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with cumulative 1 O 2 luminescence counts from PpIX in mitochondria and plasmas membrane, respectively., Results: The mitochondria are more sensitive than the plasma membrane by a factor of 1.7., Conclusions: Direct 1 O 2 luminescence dosimetry's potential value for comparing the PDT sensitivity of different subcellular organelles was demonstrated. This could be useful for developing subcellular targeted novel photosensitizers to enhance PDT efficiency., Competing Interests: Declaration of competing interest The authors have declared that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Singlet oxygen ( 1 O 2 ) is widely regarded as the main cytotoxic substance that induces the biological damage for photodynamic therapy (PDT). In this study, the previously developed near-infrared (NIR) optical imaging system was optimized for fast imaging of 1 O 2 luminescence. The optical imaging system enables direct imaging of 1 O 2 luminescence in blood vessels within 2 s during vascular-targeted PDT (V-PDT), which makes this system extremely practical for in vivo studies. The dependence of RB concentration on 1 O 2 luminescence image was investigated for V-PDT, and the data imply that 1270 nm signal is attributed to 1 O 2 luminescence. The imaging system operates with a field of view of 9.60 × 7.68 mm 2 and a spatial resolution of 30 μm, which holds the potential to elucidate the correlation between cumulative 1 O 2 luminescence and vasoconstriction for V-PDT., (© 2020 American Society for Photobiology.)

Background: Three patients enrolled in a clinical trial of 5-aminolevulinic-acid (5-ALA)-induced fluorescence-guidance, which has been demonstrated to facilitate intracranial tumor resection, were found on neuropathological examination to have focal cortical dysplasia (FCD)., Objective: To evaluate in this case series visible fluorescence and quantitative levels of protoporphyrin IX (PpIX) during surgery and correlate these findings with preoperative magnetic resonance imaging (MRI) and histopathology., Methods: Patients were administered 5-ALA (20 mg/kg) approximately 3 h prior to surgery and underwent image-guided, microsurgical resection of their MRI- and electrophysiologically identified lesions. Intraoperative visible fluorescence was evaluated using an operating microscope adapted with a commercially available blue light module. Quantitative PpIX levels were assessed using a handheld fiber-optic probe and a wide-field imaging spectrometer. Sites of fluorescence measurements were co-registered with both preoperative MRI and histopathological analysis., Results: Three patients with a pathologically confirmed diagnosis of FCD (Types 1b, 2a, and 2b) underwent surgery. All patients demonstrated some degree of visible fluorescence (faint or moderate), and all patients had quantitatively elevated concentrations of PpIX. No evidence of neoplasia was identified on histopathology, and in 1 patient, the highest concentrations of PpIX were found at a tissue site with marked gliosis but no typical histological features of FCD., Conclusion: FCD has been found to be associated with intraoperative 5-ALA-induced visible fluorescence and quantitatively confirmed elevated concentrations of the fluorophore PpIX in 3 patients. This finding suggests that there may be a role for fluorescence-guidance during surgical intervention for epilepsy-associated FCD., (Copyright © 2018 by the Congress of Neurological Surgeons.)

Background: We have developed ultrasmall porphyrin-high-density lipoprotein (HDL) nanoparticles (<20 nm), called "porphyrinHDL," that have a high density of porphyrin molecules and dissociate rapidly upon tumor cell accumulation to become fluorescent and photoactive. This is introduced as a novel activatable photosensitizer for image-guided photodynamic therapy (PDT). Here, we report the studies of these nanoparticles targeted to scavenger receptor class B type I (SR-BI) expressed on lung cancer cells as a first step toward development of a minimally invasive treatment for peripheral lung cancer and metastatic lymph nodes of advanced lung cancer., Methods: The in vitro uptake of porphyrinHDL and the corresponding PDT efficacy were evaluated in both SR-BI-positive and SR-BI-negative lung cancer cell lines. A clinically relevant orthotopic lung cancer model in mice was used to examine fluorescence activation and quantification of uptake in tumor. In addition, we investigated the effect of porphyrinHDL-mediated PDT., Results: PorphyrinHDL promoted proper intracellular uptake in the H460 human lung cancer cell line. When irradiated with a 671-nm PDT laser, porphyrinHDL produced significant therapeutic effectiveness in vitro. After systemic administration in mice with orthotopic lung cancer xenografts, porphyrinHDL demonstrated selective accumulation and photoactivation in tumor with significantly enhanced disease-to-normal tissue contrast. Moreover, porphyrinHDL-PDT significantly induced cell apoptosis in lung tumors (73.2%) without toxicity in normal tissues or damage to adjacent critical structures., Conclusions: SR-BI-targeted porphyrinHDL-mediated PDT of lung cancer is selective and effective in vitro and in vivo. These initial proof-of-principle studies suggest the potential of a "smart" PDT approach for highly selective tumor ablation., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

This paper presents a novel compact fiberoptic based singlet oxygen near-infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single-photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal-to-noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic-coupled superconducting nanowire single-photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photodynamic therapy (PDT) uses photosensitizers and visible light in combination with molecular oxygen to produce reactive oxygen species (ROS) that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature and stimulate the host immune system. The excited singlet state of oxygen ( 1 O 2 ) is recognized to be the main cytotoxic ROS generated during PDT for the majority of photosensitizers used clinically and for many investigational new agents, so that maximizing its production within tumor cells and tissues can improve the therapeutic response, and several emerging and novel approaches for this are summarized. Quantitative techniques for 1 O 2 production measurement during photosensitization are also of immense importance of value for both preclinical research and future clinical practice. In this review, emerging strategies for enhanced photosensitized 1 O 2 generation are introduced, while recent advances in direct detection and imaging of 1 O 2 luminescence are summarized. In addition, the correlation between cumulative 1 O 2 luminescence and PDT efficiency will be highlighted. Meanwhile, the validation of 1 O 2 luminescence dosimetry for PDT application is also considered. This review concludes with a discussion on future demands of 1 O 2 luminescence detection for PDT dosimetry, with particular emphasis on clinical translation. Eye-catching color image for graphical abstract., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The metastatic spread of cancer from the primary site or organ is one of its most devastating aspects, being responsible for up to 90% of cancer-associated mortality. Bone is one of the common sites of metastatic spread, including the vertebrae. Regardless of the treatment strategy, the clinical goals for patients with vertebral metastases are to improve the quality of life by preventing neurologic decline, to achieve durable pain relief and enhance local tumor control. However, in part due to the close proximity of the spinal cord, current treatment options are limited. We propose a novel therapeutic strategy with the use of photodynamic molecular beacons (PMBs) for targeted destruction of spinal metastases, particularly to de-bulk lesions as an adjuvant to vertebroplasty or kyphoplasty in order to mechanically stabilize weak or fractured vertebrae. The PDT efficacy of a matrix metalloproteinase-specific PMB is reported in a metstatic model that recapitulates the clinical features of tumor growth within the bone. We demonstrate that not only does tumor cell destruction occur but also the killing of bone stromal cells. The potential of PMB-PDT to destroy metastatic tumors, disrupt the osteolytic cycle and better preserve critical organs with an increased therapeutic window compared with conventional photosensitizers is demonstrated.

Singlet oxygen is a primary cytotoxic agent in photodynamic therapy. We show that CeF3 nanoparticles, pure as well as conjugated through electrostatic interaction with the photosensitizer verteporfin, are able to generate singlet oxygen as a result of UV light and 8 keV X-ray irradiation. The X-ray stimulated singlet oxygen quantum yield was determined to be 0.79 ± 0.05 for the conjugate with 31 verteporfin molecules per CeF3 nanoparticle, the highest conjugation level used. From this result we estimate the singlet oxygen dose generated from CeF3-verteporfin conjugates for a therapeutic dose of 60 Gy of ionizing radiation at energies of 6 MeV and 30 keV to be (1.2 ± 0.7) × 10(8) and (2.0 ± 0.1) × 10(9) singlet oxygen molecules per cell, respectively. These are comparable with cytotoxic doses of 5 × 10(7)-2 × 10(9) singlet oxygen molecules per cell reported in the literature for photodynamic therapy using light activation. We confirmed that the CeF3-VP conjugates enhanced cell killing with 6 MeV radiation. This work confirms the feasibility of using X- or γ- ray activated nanoparticle-photosensitizer conjugates, either to supplement the radiation treatment of cancer, or as an independent treatment modality.

To impart effective cellular damage via photodynamic therapy (PDT), it is vital to deliver the appropriate light dose and photosensitizer concentration, and to monitor the PDT dose delivered at the site of interest. In vivo monitoring of photosensitizers has in large part relied on their fluorescence emission. Palladium-containing photosensitizers have shown promising clinical results by demonstrating near full conversion of light to PDT activity at the cost of having undetectable fluorescence. We demonstrate that, through the coupling of plasmonic nanoparticles with palladium-photosensitizers, surface-enhanced Raman scattering (SERS) provides both reporting and monitoring capability to otherwise quiescent molecules. Nano-enabled SERS reporting of photosensitizers allows for the decoupling of the therapeutic and imaging mechanisms so that both phenomena can be optimized independently. Most importantly, the design enables the use of the same laser wavelength to stimulate both the PDT and imaging features, opening the potential for real-time dosimetry of photosensitizer concentration and PDT dose delivery by SERS monitoring.

Two-photon activated photodynamic therapy (2-γ PDT) has the potential of treating deeper tumors and/or improving tumor targeting. Here, we evaluated the one- and two-photon activated PDT efficacy of pyropheophorbide-a methyl ester (MPPa), a second-generation photosensitizer derived from chlorophyll a. We show that MPPa, when activated by femtosecond (fs) laser pulses at 674 nm, has high one-photon (1-γ) PDT efficacy against cisplatin-sensitive human cervical (HeLa) and cisplatin-resistant human lung (A549) and ovarian (NIH:OVCAR-3) cancer cells. At a low light dose of 0.06 J cm(-2), the IC50 (the MPPa concentration required to kill 50% of the cells) was determined to be 5.3 ± 0.3, 3.4 ± 0.3 and 3.6 ± 0.4 μM for HeLa, A549 and NIH:OVCAR-3 cells, respectively. More significantly, we also show that MPPa can be effectively activated by an 800 nm, 120 fs laser through 2-γ excitation; at a light dose causing no measurable photocytotoxicity in the absence of photosensitizer, the corresponding IC50 values were measured to be 4.1 ± 0.3, 9.6 ± 1.0 and 1.6 ± 0.3 μM, respectively. These results indicate that MPPa is a potent photosensitizer for both 1- and 2-γ activated PDT with potential applications for difficult-to-treat tumors by conventional therapies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Herein, we present a study of the pharmacokinetics and biodistribution of a butadiyne-linked conjugated porphyrin dimer (Oxdime) designed to have high near-infrared (NIR) 2-photon absorption cross-section for photodynamic therapy (PDT). Changes in biodistribution over time were monitored in mice carrying B16-F10 melanoma xenografts, following intravenous injection. Using fluorescence imaging of live animals and analyzing isolated organs ex vivo at different time points between 30 min and 24 h after injection, accumulation of Oxdime was measured in several organs (heart, kidney and liver) and in tumor. The concentration in the plasma was about 5-10 times higher than in other tissues. The fluorescence signal peaked at 3-12 h after injection in most tissues, including the tumor and the plasma. The change in the fluorescence emission spectrum of the sensitizer over time was also monitored and a shift in the maximum from 800 to 740 nm was observed over 24 h, showing that the Oxdime is metabolized. Significant quantities accumulated in the tumor, indicating that this PDT sensitizer may be promising for cancer treatment., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

In recent years, 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence guidance has been used as a surgical adjunct to improve the extent of resection of gliomas. Exogenous administration of ALA before surgery leads to the accumulation of red fluorescent PpIX in tumor tissue that the surgeon can visualize and thereby discriminate between normal and tumor tissue. Selective accumulation of PpIX has been linked to numerous factors, of which blood-brain barrier breakdown has been suggested to be a key factor. To test the hypothesis that PpIX concentration positively correlates with gadolinium (Gd) concentrations, we performed ex vivo measurements of PpIX and of Gd using inductively coupled plasma mass spectrometry, the latter as a quantitative biomarker of blood-brain barrier breakdown; this was corroborated with immunohistochemistry of microvascular density in surgical biopsies of patients undergoing fluorescence-guided surgery for glioma. We found positive correlations between PpIX concentration and Gd concentration (r = 0.58, p < 0.0001) and between PpIX concentration and microvascular density (r = 0.55, p < 0.0001), suggesting a significant, yet limited, association between blood-brain barrier breakdown and ALA-induced PpIX fluorescence. To our knowledge, this is the first time that Gd measurements by inductively coupled plasma mass spectrometry have been used in human gliomas.

5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence can be used to identify tissue for resection using an adapted operating microscope. A multi-institutional clinical trial comparing fluorescence-guided versus white light tumor resection reported significant improvement in completeness of resection and 6-month progression-free survival. The degree of 5-ALA-induced fluorescence correlates with histopathologic grade of tumor, degree of tumor cell infiltration, and proliferation indices. Quantitative methodologies for assessment of tissue fluorescence have significantly improved the ability to detect tumor tissue and intraoperative diagnostic performance. These developments extend the applicability of this technology to additional tumor histologies and provide the rationale for further instrumentation development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen ((1)O(2)) through interactions of photosensitizer, light, and oxygen ((3)O(2)). These three components are highly interdependent and dynamic, resulting in variable temporal and spatial (1)O(2) dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from (1)O(2) provides a direct and predictive PDT dose metric, it may not be clinically practical. We used (1)O(2) luminescence (or singlet oxygen luminescence (SOL)) as a gold-standard metric to evaluate potentially more clinically feasible dosimetry based on photosensitizer bleaching. We performed in vitro dose-response studies with simultaneous SOL and photosensitizer fluorescence measurements under various conditions, including variable (3)O(2), using the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC). The results show that SOL was always predictive of cytotoxicity and immune to PDT's complex dynamics, whereas photobleaching-based dosimetry failed under hypoxic conditions. However, we identified a previously unreported 613 nm emission from mTHPC that indicates critically low (3)O(2) levels and can be used to salvage photobleaching-based dosimetry. These studies improve our understanding of PDT processes, demonstrate that SOL is a valuable gold-standard dose metric, and show that when used judiciously, photobleaching can serve as a surrogate for (1)O(2) dose., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Object: Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative fluorescence of protoporphyrin IX (PpIX), synthesized endogenously following δ-aminolevulinic acid (ALA) administration, has been used for this purpose in high-grade glioma (HGG). The authors show that diagnostically significant but visually imperceptible concentrations of PpIX can be quantitatively measured in vivo and used to discriminate normal from neoplastic brain tissue across a range of tumor histologies., Methods: The authors studied 14 patients with diagnoses of low-grade glioma (LGG), HGG, meningioma, and metastasis under an institutional review board-approved protocol for fluorescence-guided resection. The primary aim of the study was to compare the diagnostic capabilities of a highly sensitive, spectrally resolved quantitative fluorescence approach to conventional fluorescence imaging for detection of neoplastic tissue in vivo., Results: A significant difference in the quantitative measurements of PpIX concentration occurred in all tumor groups compared with normal brain tissue. Receiver operating characteristic (ROC) curve analysis of PpIX concentration as a diagnostic variable for detection of neoplastic tissue yielded a classification efficiency of 87% (AUC = 0.95, specificity = 92%, sensitivity = 84%) compared with 66% (AUC = 0.73, specificity = 100%, sensitivity = 47%) for conventional fluorescence imaging (p < 0.0001). More than 81% (57 of 70) of the quantitative fluorescence measurements that were below the threshold of the surgeon's visual perception were classified correctly in an analysis of all tumors., Conclusions: These findings are clinically profound because they demonstrate that ALA-induced PpIX is a targeting biomarker for a variety of intracranial tumors beyond HGGs. This study is the first to measure quantitative ALA-induced PpIX concentrations in vivo, and the results have broad implications for guidance during resection of intracranial tumors.

Breast cancer is the second leading cause of cancer-related death in women. Approximately 85% of patients with advanced cases will develop spinal metastases. The vertebral column is the most common site of breast cancer metastases, where overexpression of matrix metalloproteinases (MMPs) promotes the spread of cancer. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. An attractive alternative is photodynamic therapy providing noninvasive and site-selective treatment. However, current photosensitizers are limited by their nonspecific accumulation. Photodynamic molecular beacons (PP(MMP)B), activated by MMPs, offer another level of PDT selectivity and image-guidance preserving criticial tissues, specifically the spinal cord. Metastatic human breast carcinoma cells, MT-1, were used to model the metastatic behavior of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PP(MMP)B in MT-1 cells. Using a clinically relevant metastatic model, fluorescent imaging establishes the specific activation of PP(MMP)B by vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of these beacons. Here, we validate that the metastasis-selective mechanism of PP(MMP)Bs can specifically image breast cancer vertebral metastases, thereby differentiating tumor and healthy tissue.

Object: Complete resection of skull base meningiomas provides patients with the best chance for a cure; however, surgery is frequently difficult given the proximity of lesions to vital structures, such as cranial nerves, major vessels, and venous sinuses. Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative assessment of protoporphyrin IX (PpIX) fluorescence following the exogenous administration of 5-aminolevulinic acid (ALA) has demonstrated utility in malignant glioma resection but limited use in meningiomas. Here the authors demonstrate the use of ALA-induced PpIX fluorescence guidance in resecting a skull base meningioma and elaborate on the advantages and disadvantages provided by both quantitative and qualitative fluorescence methodologies in skull base meningioma resection., Methods: A 52-year-old patient with a sphenoid wing WHO Grade I meningioma underwent tumor resection as part of an institutional review board-approved prospective study of fluorescence-guided resection. A surgical microscope modified for fluorescence imaging was used for the qualitative assessment of visible fluorescence, and an intraoperative probe for in situ fluorescence detection was utilized for quantitative measurements of PpIX. The authors assessed the detection capabilities of both the qualitative and quantitative fluorescence approaches., Results: The patient harboring a sphenoid wing meningioma with intraorbital extension underwent radical resection of the tumor with both visibly and nonvisibly fluorescent regions. The patient underwent a complete resection without any complications. Some areas of the tumor demonstrated visible fluorescence. The quantitative probe detected neoplastic tissue better than the qualitative modified surgical microscope. The intraoperative probe was particularly useful in areas that did not reveal visible fluorescence, and tissue from these areas was confirmed as tumor following histopathological analysis., Conclusions: Fluorescence-guided resection may be a useful adjunct in the resection of skull base meningiomas. The use of a quantitative intraoperative probe to detect PpIX concentration allows more accurate determination of neoplastic tissue in meningiomas than visible fluorescence and is readily applicable in areas, such as the skull base, where complete resection is critical but difficult because of the vital structures surrounding the pathology.

To date, singlet oxygen ((1)O(2)) luminescence (SOL) detection was predictive of photodynamic therapy (PDT) treatment responses both in vitro and in vivo, but accurate quantification is challenging. In particular, the early and strongest part of the time-resolved signal (500-2000ns) is difficult to separate from confounding sources of luminescence and system noise, and so is normally gated out. However, the signal dynamics change with oxygen depletion during PDT, so that this time gating biases the (1)O(2) measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO(2) measurements during meso-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated PDT of cells in vitro under well-controlled conditions. With these data as input, numerical simulations were used to examine PDT and SOL dynamics, and the influence of various time gates on cumulative SOL signals. It is shown that gating can underestimate the SOL at early treatment time points by ∼40% and underestimate the cumulative SOL signal by 20-25%, representing significant errors. In vitro studies with both mTHPC and aminolevulinic acid-photosensitizer protoporphyrin IX demonstrate that rigorous analysis of SOL signal kinetics is then crucial in order to use SOL as an accurate and quantitative PDT dose metric., (© 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.)

Breast cancer patients commonly develop metastases in the spine, which compromises its mechanical stability and can lead to skeletal related events. The current clinical standard of treatment includes the administration of systemic bisphosphonates (BP) to reduce metastatically induced bone destruction. However, response to BPs can vary both within and between patients, which motivates the need for additional treatment options for spinal metastasis. Photodynamic therapy (PDT) has been shown to be effective at treating metastatic lesions secondary to breast cancer in an athymic rat model, and is proposed as a treatment for spinal metastasis. The objective of this study was to determine the effect of PDT, alone or in combination with previously administered systemic BPs, on the structural and mechanical integrity of both healthy and metastatically involved vertebrae. Human breast carcinoma cells (MT-1) were inoculated into athymic rats (day 0). At 14 days, a single PDT treatment was administered, with and without previous BP treatment at day 7. In addition to causing tumor necrosis in metastatically involved vertebrae, PDT significantly reduced bone loss, resulting in strengthening of the vertebrae compared to untreated controls. Combined treatment with BP + PDT further enhanced bone architecture and strength in both metastatically involved and healthy bone. Overall, the ability of PDT to both ablate malignant tissue and improve the structural integrity of vertebral bone motivates its consideration as a local minimally invasive treatment for spinal metastasis secondary to breast cancer.

Nucleic acid photodynamic molecular beacons (PMBs) are a class of activatable photosensitizers that increase singlet oxygen generation upon binding a specific target sequence. Normally, PMBs are functionalized with multiple solution-phase labeling and purification steps. Here, we make use of a flexible solid-phase approach for completely automated synthesis of PMBs. This enabled the creation of a new type of molecular beacon that uses a linear superquencher architecture. The 3' terminus was labeled with a photosensitizer by generating pyropheophorbide-labeled solid-phase support. The 5' terminus was labeled with up to three consecutive additions of a dark quencher phosphoramidite. These photosensitizing and quenching moieties were stable in the harsh DNA synthesis environment and their hydrophobicity facilitated PMB purification by HPLC. Linear superquenchers exhibited highly efficient quenching. This fully automated synthesis method simplifies not only the synthesis and purification of PMBs, but also the creation of new activatable photosensitizer designs.

Breast cancer is known to cause metastatic lesions in the bone, which can lead to skeletal-related events. Currently, radiation therapy and surgery are the treatment of choice, but the success rate varies and additional adjuncts are desirable. Photodynamic therapy (PDT) has been applied successfully as a non-radiative treatment for numerous cancers. Earlier work has shown that the athymic rat model is suitable to investigate the effect of PDT on bone metastasis and benzoporphyrin-derivative monoacid ring A (BPD-MA; verteporfin) has been shown to be a selective photosensitizer. The aim of this study was to define the therapeutic window of photosensitizer with regard to drug and light dose. Human breast carcinoma cells (MT-1)-stable transfected with the luciferase gene-were injected intra-cardiacally into athymic rats. At 14 days, the largest vertebral lesion by bioluminescence imaging was targeted for single treatment PDT. A drug escalating-de-escalating scheme was used (starting drug dose and light energy of 0.2 mg/kg and 50 J, respectively). Outcomes included 48 h post-treatment bioluminescence of remaining viable tumour, histomorphometric assessment of tumour burden, and neurologic evaluation. The region of effect by bioluminescence and histology increased with increasing drug dose and light energy. A safe and effective drug-light dose combination in this model appears to be 0.5 mg/kg BPD-MA and applied light energy of less than 50 J for the thoracic spine and 1.0 mg/kg and 75 J for the lumbar spine. For translation to clinical use, it is an advantage that BPD-MA (verteporfin), a second-generation photosensitizer, is already approved to treat age-related macular degeneration. Overall, PDT represents an exciting potential new minimally-invasive local, safe and effective therapy in the management of patients with spinal metastases.

As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.

Photodynamic therapy uses nonthermal coherent light delivered via fiber optic cable to locally activate a photosensitive chemotherapeutic agent that ablates tumor tissue. Owing to the limitations of light penetration, it is unknown whether photodynamic therapy can treat large osseous tumors. We determined whether photodynamic therapy can induce necrosis in large osseous tumors, and if so, to quantify the volume of treated tissue. In a pilot study we treated seven dogs with spontaneous osteosarcomas of the distal radius. Tumors were imaged with MRI before and 48 hours after treatment, and the volumes of hypointense regions were compared. The treated limbs were amputated immediately after imaging at 48 hours and sectioned corresponding to the MR axial images. We identified tumor necrosis histologically; the regions of necrosis corresponded anatomically to hypointense tissue on MRI. The mean volume of necrotic tissue seen on MRI after photodynamic therapy was 21,305 mm(3) compared with a pretreatment volume of 6108 mm(3). These pilot data suggest photodynamic therapy penetrates relatively large canine osseous tumors and may be a useful adjunct for treatment of bone tumors.

The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-alpha (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J > or = 5.1 x 10(13) M(-1) cm(-1) nm4) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J > or = 3.7 x 10(15) M(-1) cm(-1) nm4) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.

We report a new class of photodynamic molecular beacon (PMB) with tumor specific mRNA-triggered control of singlet oxygen ((1)O(2)) production. The beacon contains a single-stranded oligonucleotide linker that forms a stem-loop structure (hairpin) in which the sequence is an antisense oligonucleotide (AS-ON) complementary to a target mRNA. The stem is formed by the annealing of two complementary arm sequences that are on either side of the loop sequence. A photosensitizer molecule (PS) is attached to the end of one arm and a quencher (Q) is similarly attached to the other end. The conformationally-restricted hairpin forces Q to efficiently silence the photoreactivity of PS. In the presence of target mRNA, the hairpin opens and the PS is no longer silenced. Upon irradiating with light, the PS then emits fluorescence and generates cytotoxic (1)O(2). To show proof of concept, we have synthesized a c-raf-1 mRNA-triggered PMB using pyropheophorbide (Pyro) as PS, carotenoid as Q and c-raf-1 mRNA-targeted AS-ON as the loop sequence. We show that the (1)O(2) production of Pyro is quenched in its native state by 15-fold and is restored 9-fold by the addition of the target RNA. Comparing this to our recently reported self-folding peptide linker-based PMB, the hairpin effect results in an enhanced (1)O(2) quenching efficiency that decreases the residual (1)O(2) production by over 3-fold, thus providing enhanced control of (1)O(2) production upon target-linker interactions. When incubated with c-raf-1 expressing MDA-MB-231 cancer cells, the PMB displayed efficient cellular uptake and subsequently effective PDT activation in targeted cells.

Photodynamic therapy (PDT) mediated with vascular acting photosensitizer Tookad (Pd-bacteriopheophorbide) was investigated as an alternative modality for treating prostate cancer. Photodynamic effects on the prostate gland and its adjacent tissues were evaluated in a canine model. Interstitial prostate PDT was performed by irradiating individual lobes with a cylindrical diffuser fiber at various drug/light doses. The sensitivity of the adjacent tissues to Tookad PDT was determined by directly irradiating the surface of the bladder, colon, abdominal muscle and pelvic plexus with a microlens fiber at various drug/light doses. The prostate and adjacent tissues were harvested one-week after the treatment and subjected to histopathological examination. PDT-induced prostate lesions were characterized by marked hemorrhagic necrosis. The bladder, colon, abdominal muscle and pelvic plexus appeared to be sensitive to PDT although the Tookad PDT-induced responses in these tissues were minimal compared to that of the prostate gland at the same dose levels. Nevertheless, the protection of the adjacent tissues should be taken into consideration during the total prostate ablation process due to their sensitivity to PDT. The sensitivity of the prostatic urethra is worth further investigation. Direct intraurethral irradiation might provide an ideal means to determine the sensitivity of the prostatic urethra and might lead to transurethral PDT protocols for the management of benign prostatic hyperplasia (BHP).

Molecular beacons are FRET-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets, thus are useful tools for in vivo cancer imaging. Photodynamic therapy (PDT) is a cell-killing process by light activation of a photosensitizer (PS) in the presence of oxygen. The key cytotoxic agent is singlet oxygen ((1)O(2)). By combining these two principles (FRET and PDT), we have introduced a concept of photodynamic molecular beacons (PMB) for controlling the PS's ability to generate (1)O(2) and, ultimately, for controlling its PDT activity. The PMB comprises a disease-specific linker, a PS, and a (1)O(2) quencher, so that the PS's photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease. Here, we report the full implementation of this concept by synthesizing a matrix metalloproteinase-7 (MMP7)-triggered PMB and achieving not only MMP7-triggered production of (1)O(2) in solution but also MMP7-mediated photodynamic cytotoxicity in cancer cells. Preliminary in vivo studies also reveal the MMP7-activated PDT efficacy of this PMB. This study validates the core principle of the PMB concept that selective PDT-induced cell death can be achieved by exerting precise control of the PS's ability to produce (1)O(2) by responding to specific cancer-associated biomarkers. Thus, PDT selectivity will no longer depend solely on how selectively the PS can be delivered to cancer cells. Rather, it will depend on how selective a biomarker is to cancer cells, and how selective the interaction of PMB is to this biomarker.

Background and Objective: To determine the optimal magnetic resonance imaging (MRI) methodology to assess photodynamic therapy (PDT)-induced histopathological responses in the prostate., Study Design/materials and Methods: Laparotomy was performed in five healthy dogs. Cylindrical diffuser was placed in the prostates to deliver light of 50-300 J/cm at 150 mW/cm and 763 nm to activate IV-injected Tookad (1 mg/kg b.w.). Fast spin echo (FSE) T2-weighted, post-contrast-enhanced T1-(CE-T1) and diffusion weighted images (DWI) were obtained pre- and 2 days, 7 days, and 1 month post-PDT. Radiological-histopathological correlation was performed at 7 days (n = 4) and 1 month (n = 1) after PDT. A qualitative assessment of signal changes and apparent diffusion coefficient (ADC) mapping was performed., Results: At 2 or 7 days post-PDT, there was good spatial correlation between PDT-induced hemorrhagic necrosis and unenhanced regions on CE-T1 images. There was a rapidly and persistently enhancing rim corresponding to edema and inflammation. FSE T2 and DWI showed altered signal but did not clearly define necrosis in all cases. At 1 month, it was hard to correlate MR images to histopathologic changes as they represented a mixture of necrosis and developing fibrosis, which led to a mixed signal intensity and less demarcated contrast enhancement., Conclusions: At 7 days after PDT, gadolinium DTPA contrast-enhanced MRI is superior to DWI and T2 imaging in assessing the boundary of Tookad PDT-induced tissue necrosis in the normal canine prostate., (Copyright 2006 Wiley-Liss, Inc.)

Background and Objectives: PDT has been used in the treatment of malignant brain tumors. This communication updates our series of unselected malignant gliomas treated with Photofrin-PDT., Study Design and Methods: We examined the records of 112 patients with malignant gliomas, metastatic brain tumors and meningiomas treated with Photofrin-PDT at St. Michael's Hospital, Toronto. These patients were treated prior to the onset of ongoing randomized trials in Photofrin PDT or had pathology that excluded them from such trials., Results: The overall post-PDT survival of 96 patients with supratentorial gliomas was 42 weeks, with a 40 and 22% 1- and 2-year survival, respectively. The greater the degree of oligodendroglial involvement the longer was the survival. Seventy-five percent of patients had no significant post-operative complications., Conclusions: Photofrin-PDT was safe. However, higher light doses than were used in these patients may be required for improved responses., (Copyright 2006 Wiley-Liss, Inc.)

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-gamma) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-gamma) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-gamma excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-gamma PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-gamma killing of vascular endothelial cells in vitro. The 2-gamma absorption properties of Photofrin were investigated in the 750-900 nm excitation wavelength range. It was shown that 2-gamma excitation dominates over 1-gamma excitation above 800 nm. The 2-gamma absorption spectrum of Photofrin in the 800-900 nm excitation wavelength range was measured. The 2-gamma cross section decreased from about 10 GM (1 GM = 10(-50) cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-gamma contribution was negligible. Cell death was monitored with the use of 2-gamma scanning laser microscopy. The light doses required for killing were high (6300 J cm(-2) for approximately 50% killing), but 2-gamma cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-gamma PDT due to its low 2-gamma cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-gamma cross sections (>100 GM), which will be required for 2-gamma PDT of AMD (and other conditions) to be clinically practical.

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 1000 neonates and 1 in 5000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regimens 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 (S. aureus) grown onto kirschner wires (K-wire). S. aureus-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 S. aureus biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. S. aureus infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg kg(-1)) intraperitoneally followed 4 h later by light (635 +/- 10 nm; 75 J cm(-2)) delivered transcutaneously via an optical fiber placed onto the tibia and resulted in significant delay in bacterial growth. In vitro, MB and ALA displayed similar cell kill with > or =4 log(10) 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 S. aureus cultures grown in vitro or in vivo using an animal model of osteomyelitis.

Background and Objectives: Photodynamic therapy (PDT) mediated with Tookad (Pd-bacteriopheophorbide, WST09) was investigated pre-clinically as part of a program to develop an alternative modality for treating prostate cancer., Study Design/materials and Methods: Spontaneous canine prostate cancer and normal canine prostate were used as the animal models. Interstitial PDT was performed by IV infusion of the photosensitizer and irradiating the prostates with a diode laser (763 nm). The prostates were harvested 1-week post-PDT and subjected to histopathologic examinations. The effects of the drug doses and light doses were studied for one- and two-session PDT. Pharmacokinetics were studied using HPLC assay. The feasibility of using perfusing CT scans for assessing PDT lesions was also evaluated., Results: Tookad is a vascular-acting drug and clears rapidly from the circulation. Tookad-PDT-induced lesions, in both normal and cancerous prostates, were characterized by marked hemorrhagic necrosis., Conclusions: Tookad-PDT is very effective in ablating prostatic tissue through its vascular effects., (Copyright 2005 Wiley-Liss, Inc.)

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.

TOOKAD (WST09) is a new, long-wavelength palladium bacteriopheophorbide photosensitizer that targets tissue vasculature. The cutaneous phototoxicity of TOOKAD was assessed in normal rat and pig animal models and in patients in a Phase-I trial of TOOKAD-mediated photodynamic therapy (PDT) for recurrent prostate cancer. Controlled skin exposures were administered using solar-simulated light at various times after drug administration. Two different spectral ranges were used. In the first, the UV portion of the spectrum was removed (UV(-)) because UV irradiation in nondrugged control animals produced an erythema response at incident energy densities (J/cm(2)) lower than those required to induce a PDT response. In the second, the full solar spectrum (UV(+)) was used, and the potentiation by the photosensitizer of the UV-mediated minimum erythema dose was assessed. Results showed that the PDT skin response was negligible at clinical drug doses of 2 mg/kg for any period after administration at light doses of 128 J/cm(2) in the animal models. In patients, there was no observed UV(-) skin response at doses of up to 2 mg/kg, drug-light intervals of 1-3 h or greater and light exposures up to 128 J/cm(2). At higher drug doses in the rat and pig models, the duration of skin phototoxicity was found to be approximately 3 h and less than 1 h, respectively. Using the full spectrum of solar-simulated light, the presence of TOOKAD did not measurably enhance the UV(+)-induced erythema in the rats, pigs or patients.

We report a new concept for type-II photosensitization, based on incorporating the photosensitizer (PS) and a singlet-oxygen (1O2) quenching/scavenging molecule onto a disease-targeting linker, such that the PS becomes activatable by light only when targeting has occurred. In this first proof-of-concept report, a model photosensitizing beacon was synthesized containing a pyropheophorbide as the PS and a carotenoid as the 1O2 quencher. These were kept in close proximity by the self-folding of a caspase-3-specific peptide sequence. Upon caspase-3-induced cleavage, the 1O2 production increased markedly, as measured directly by 1O2 near-infrared luminescence and lifetime measurements.

Photodynamic therapy (PDT) is a promising modality for the treatment of solid tumors that combines a photosensitizing agent and light to produce cytotoxic reactive oxygen species that lead to tumor cell death. The recent introduction of bioluminescence imaging (BLI), involving the use of the luciferase gene (luc) transferred into target tumor cells, followed by systemic administration of luciferin and detection of the emitted visible chemiluminescence photons, offers the potential for longitudinal imaging of tumor growth and therapeutic response in single animals. We demonstrate in this study the first results of the use of BLI to assess the response of an intracranial brain tumor model (9L rat gliosarcoma) to aminolevulinic acid (ALA)-mediated PDT. Complementary in vitro experiments with the luciferase-transfected 9L cells show that the decrease in the luminescent signal after PDT correlates with cell kill. In vivo imaging shows a decrease in the BLI signal from the tumor after ALA-PDT treatment, followed by tumor regrowth. Furthermore, preliminary studies using cells transfected with a hypoxia-responsive vector show an increase in bioluminescence within 4 h after Photofrin-mediated PDT, demonstrating the ability to observe stress-gene responses. These results suggest that BLI can be used to provide spatiotemporal information of intracranial brain tumor responses after PDT and may serve as a valuable response-endpoint measure.

Photodynamic therapy (PDT) uses light to activate a photosensitizer to achieve localized tumor control. In this study, PDT mediated by a second-generation photosensitizer, palladium-bacteriopheophorbide WST09 (Tookad) was investigated as an alternative therapy for prostate cancer. Normal canine prostate was used as the animal model. PDT was performed by irradiating the surgically exposed prostate superficially or interstitially at 763 nm to different total fluences (100 or 200 J/cm2; 50, 100 or 200 J/cm) at 5 or 15 min after intravenous administration of the drug (2 mg/kg). Areas on the bladder and colon were also irradiated. The local light fluence rate and temperature were monitored by interstitial probes in the prostate. All animals recovered well, without urethral complications. During the 1 week to 3 month post-treatment period, the prostates were harvested for histopathological examination. The PDT-induced lesions showed uniform hemorrhagic necrosis and atrophy, were well delineated from the adjacent normal tissue and increased linearly in diameter with the logarithm of the delivered light fluence. A maximum PDT-induced lesion size of over 3 cm diameter could be achieved with a single interstitial treatment. There was no damage to the bladder or rectum caused by scattered light from the prostate. The bladder and rectum were also directly irradiated with PDT. At 80 J/cm2, a full-depth necrosis was observed but resulted in no perforation. At 40 J/cm2, PDT produced minimal damage to the bladder or rectum. On the basis of optical dosimetry, we have estimated that 20 J/cm2 is the fluence required to produce prostatic necrosis. Thus, the normal structure adjacent to the prostate can be safely preserved with careful dosimetry. At therapeutic PDT levels, there was no structural or functional urethral damage even when the urethra was within the treated region. Hence, Tookad-PDT appears to be a promising candidate for prostate ablation in patients with recurrent, or possibly even primary, prostate cancer.

The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET(2)), and meta -tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer., (Copyright 2000 Cancer Research Campaign.)

The photodynamic threshold dose in normal rat liver was determined from the measured depth of necrosis following surface irradiation. The threshold was determined for the photosensitizing drugs Photofrin and monosulfonated aluminum chlorophthalocyanine, AlPcS1, at 24 h postinjection and was found to be (3.4 x/divided by 1.3) x 10(18) and (8.2 x/divided by 1.5) x 10(18) photons cm-3, respectively, compared with the previously reported value of (38 +/- 2) x 10(18) photons cm-3 for the tri/tetrasulfonated phthalocyanine, AlPcS4. These values were independent of drug concentration or total light fluence. For all three drugs the depth of tissue necrosis decreased as the time between drug and light administration increased from 10 min to 72 h. This decrease can be attributed both to the change in the tissue drug concentration as well as to changes in the efficiency of photodynamic therapy for producing tissue damage, related to the photodynamic necrosis threshold. The threshold values for all three photosensitizers were lowest at 10 min post injection: (1.4 x/divided by 1.4) x 10(18), (1.6 x/divided by 1.3) x 10(18) and (23 x/divided by 1.3) x 10(18) photons cm-3 for Photofrin, AlPcS1 and AlPcS4, respectively. The changes in necrosis threshold with time may be due to an initial change from entirely vascular to a combination of vascular and cellular damage, with later redistribution of the photosensitizer to targets at the subcellular level.

Few studies have been published to date measuring spatially resolved fluence rates in complex tissue geometries. Here the light distributions of three different intraperitoneal light delivery geometries in a murine ovarian cancer model were investigated to assess their influence on the tumorcidal efficacy of photodynamic therapy (PDT). In vivo fluence rate measurements in the peritoneal cavities of mice, with the light intensity being mapped in three transverse planes, were performed using fiber-optic detectors. Three different source fiber designs and placements were tested for their ability to provide uniform irradiation of the peritoneal cavity. The biological response to a PDT protocol comprising three separate treatments administered at 72 h intervals, each consisting of a 0.25 mg kg-1 intraperitoneal injection of benzoporphyrin derivative-mono acid ring A followed 90 min later by delivery of 15 J of 690 nm light, was measured. The tissue response was evaluated by measuring the number of remaining visible lesions and the total residual tumor mass. Fluence rate measurements showed large variations in the fluence rate distribution for similar intended treatments. The most uniform and reproducible illumination was achieved using two 18 mm long cylindrical emitting optical fibers. The biological response was comparable to that produced when a flat-cleaved end optical fiber is used to illuminate the four quadrants of the abdomen sequentially. While a good reproducibility in tumor induction in this animal model exists, no correlation was found between the fluence rate distribution measured in one group of animals and the biological response in a separate group of similarly treated animals. Due to the large intra-animal variability in fluence rate distribution, representative fluence rate mapping in complex tissue geometries is of limited value when applied to an individual PDT treatment. Thus, surveillance of the fluence rate during individual treatments will be required for acceptable PDT dosimetry. To improve the versatility of this particular animal model for PDT research, a large number of extended sources are required to increase uniformity of the illumination in order to reduce unwanted cytotoxic side effects resulting from foci of high fluence rates. In this way, subsequent increase of the total energy delivered to the tumor may be possible.

Objective: The effectiveness of four different photosensitizers for intracranial photodynamic therapy (PDT) of normal brain tissues and an intracranial tumor was investigated in rabbits, using the photodynamic threshold model., Summary: PDT is currently being investigated as an adjuvant treatment to surgical resection and/or radio chemotherapy of intracranial neoplasms. While possible neurotoxic side effects of the treatment have been noted, only limited preclinical data quantifying the response of intracranial normal and tumor tissues following PDT are available., Materials and Methods: The photodynamic threshold dose values for the four photosensitizers, Photofrin, 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX), Tin Ethyl Etiopurpurin (SnET2), and chloroaluminum phthalocyanine (AlClPc), were determined using measured light fluence distributions, photosensitizer concentration in tissue, and histologically-determined extent of necrosis following PDT. These measurements were made in normal rabbit brain and in an intracranially-implanted carcinoma (VX2)., Results: For Photofrin, AlClPc, and SnET2 (in an emulsion delivery vehicle) normal grey and white matter were very sensitive to PDT, showing a significantly lower threshold dose value than VX2-tumor. For ALA-induced PpIX and SnET2 (in liposome) very little or no white matter damage was observed. Additionally, ALA-PpIX showed significantly lower concentration in white matter than in cortex and tumor. Normal brain structures lacking a blood-brain barrier showed high uptake of all photosensitizers and, hence, are at risk of collateral damage during PDT., Conclusions: For clinical PDT of most adult intracranial neoplasms ALA-induced PpIX appears to be promising, and SnET2 (liposomal) has potential for selective tumor destruction with relative sparing of white matter. Other normal brain structures and, for the other photosensitizers, also white matter are at risk of collateral damage, if exposed to light during PDT.

The determination of the photosensitizer concentration in ex vivo tissue samples is commonly used for pharmacokinetic and dosimetric studies of photodynamic therapy, both clinically and pre-clinically. In this report, a new method is presented based on tissue solubilization and subsequent fluorometry. This method has the advantages of good sensitivity, accuracy and reproducibility, as well as low cost and ease of handling of the tissue samples. The method was tested for six different photosensitizers in a variety of tissues. The accuracy and concentration detection limits are compared with those of other published extraction methods.

The applicability and limitations of a photodynamic threshold model, used to describe quantitatively the in vivo response of tissues to photodynamic therapy, are currently being investigated in a variety of normal and malignant tumour tissues. The model states that tissue necrosis occurs when the number of photons absorbed by the photosensitiser per unit tissue volume exceeds a threshold. New Zealand White rabbits were sensitised with porphyrin-based photosensitisers. Normal brain or intracranially implanted VX2 tumours were illuminated via an optical fibre placed into the tissue at craniotomy. The light fluence distribution in the tissue was measured by multiple interstitial optical fibre detectors. The tissue concentration of the photosensitiser was determined post mortem by absorption spectroscopy. The derived photodynamic threshold values for normal brain are significantly lower than for VX2 tumour for all photosensitisers examined. Neuronal damage is evident beyond the zone of frank necrosis. For Photofrin the threshold decreases with time delay between photosensitiser administration and light treatment. No significant difference in threshold is found between Photofrin and haematoporphyrin derivative. The threshold in normal brain (grey matter) is lowest for sensitisation by 5 delta-aminolaevulinic acid. The results confirm the very high sensitivity of normal brain to porphyrin photodynamic therapy and show the importance of in situ light fluence monitoring during photodynamic irradiation.