This PDF file contains the front matter associated with SPIE Proceedings Volume 7886, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

While many of the determinants of photodynamic tumor eradication have been identified, the story is not yet complete. Fluorescent probes for reactive oxygen species (ROS) are seldom specific, and the role of different ROS in apoptosis vs. autophagy are not fully delineated. Moreover, the conflicting roles of autophagy as both a death and a survival pathway remain to be explained. Most tissue-culture studies are carried out in 20% oxygen although this is far in excess of the environment of malignant cells in vivo. And while apoptotic and/or autophagic death appears to account for the lethal effects of PDT, an effect on membrane recycling has now been identified. In this report, we summarize some recent experiments designed to examine the specificity of fluorescent ROS probes. We also demonstrate the ability of hydrogen peroxide to accelerate the autophagic response to PDT in an adhering cell line, the 1c1c7 murine hepatoma. In this cell line, autophagy appears to be a pro-survival mechanism since a sub-line (KD) depleted in a critical autophagy protein (atg7) was more responsive to PDT than wild-type (WT) cells. There are clearly multiple determinants of direct tumor cell kill by PDT that depend on the PDT target, the ROS produced and phenotypic variations.

Three-dimensional in vitro tumor models have emerged as powerful research tools in cancer biology, though the vast potential of these systems as high-throughput, biologically relevant reporters of treatment response has yet to be adequately explored. Here, building on previous studies, we demonstrate the utility of using 3D models for ovarian and pancreatic cancers in conjunction with quantitative image processing to reveal aspects of growth behavior and treatment response that would not be evident without either modeling or quantitative analysis component. In this report we specifically focus on recent improvements in the imaging component of this integrative research platform and emphasize analysis to establish reproducible growth properties in 3D tumor arrays, a key consideration in establishing the utility of this platform as a reliable reporter of therapeutic response. Building on previous studies using automated segmentation of low magnification image fields containing large numbers of nodules to study size dependent treatment effects, we introduce an improvement to this method using multiresolution decomposition to remove gradient background from transmitted light images for more reliable feature identification. This approach facilitates the development of a new treatment response metric, disruption fraction (Dfrac), which quantifies dose dependent distribution shifts from nodular fragmentation induced by cytotoxic therapies. Using this approach we show that PDT treatment is associated with significant dose-dependent increases in Dfrac, while this is not observed with carboplatin treatment. The ability to quantify this response to therapy could play a key role in design of combination regimens involving these two modalities.

The development and translational potential of therapeutic strategies for cancer is limited, in part, by a lack of biological models that capture important aspects of tumor growth and treatment response. It is also becoming increasingly evident that no single treatment will be curative for this complex disease. Rationally-designed combination regimens that impact multiple targets provide the best hope of significantly improving clinical outcomes for cancer patients. Rapidly identifying treatments that cooperatively enhance treatment efficacy from the vast library of candidate interventions is not feasible, however, with current systems. There is a vital, unmet need to create cell-based research platforms that more accurately mimic the complex biology of human tumors than monolayer cultures, while providing the ability to screen therapeutic combinations more rapidly than animal models. We have developed a highly reproducible in vitro three-dimensional (3D) tumor model for micrometastatic ovarian cancer (OvCa), which in conjunction with quantitative image analysis routines to batch-process large datasets, serves as a high throughput reporter to screen rationally-designed combination regimens. We use this system to assess mechanism-based combination regimens with photodynamic therapy (PDT), which sensitizes OvCa to chemo and biologic agents, and has shown promise in clinic trials. We show that PDT synergistically enhances carboplatin efficacy in a sequence dependent manner. In printed heterocellular cultures we demonstrate that proximity of fibroblasts enhances 3D tumor growth and investigate co-cultures with endothelial cells. The principles described here could inform the design and evaluation of mechanism-based therapeutic options for a broad spectrum of metastatic solid tumors.

A three-compartment kinetic model for the binding of a ligand to its receptor in tumor tissue has been explained and the kinetic rates of the model are currently being investigated. In order to determine the plasma excretion rates of the dyes of interest, the fluorescence extinction coefficients must be determined. The fluorescence extinction coefficients of the IRDye700DX-carboxylate (IRDye700DX-C) and IRDye800CW-conjugated to EGFR (IRDye800CW-EGF) have been to be 7.98 ×10⁶ μM^-1 cm^-1 and 4.73x10⁶ μM^-1 cm^-1, respectively. We determined that the linear range of these dyes in the blood plasma of a mouse was 0 - 0.26 μM. Administration of 1 nmol of each of these dyes to a mouse weighing 25-30g (0.04 μM - 0.033 μM, respectively) will result in blood plasma fluorescence in the linear and readable range.

The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell death, although the time course is slower compared to Pc 4-PDT. In A431 epidermoid carcinoma cells, these new analogues preferentially localized in lysosomes were highly efficient in inducing apoptotic cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced mitochondrial depolarization and cell killing. Both depolarization and cell killing were substantially prevented by iron chelators. The fact that Pc 4-PDT plus bafilomycin treatment did not induce lysosomal membrane damage prior to mitochondrial depolarization suggests that bafilomycin instead induced release of redox active iron from lysosomes into the cytosol that further translocated into mitochondria, where iron-mediated free radical formation occurred. In conclusion, agents that disturb lysosomal function could potentially be used as adjuvants with mitochondrion-targeted photosensitizers to enhance phototoxicity.

Folate receptors are over expressed in many types of cancers, including, ovarian, breast, and cervical. In our continuing efforts toward the development of targeted Type 1 phototherapeutic agents, an azide-based Type 1 photosensitizer and a pyrzine-based fluorophore that absorb and emits in the visible region, and a dual diagnostic-therapeutic probe consisting of the fluorophore and the photosensitizer were prepared and independently conjugated to two folate receptor specific vectors: γ-carboxyl-modified folic acid and anti-human FOLR1 (folate receptor-1) antibody In vitro receptor binding study showed that all the conjugates had high (ca 1-7 nM) affinity to the folate receptor. Confocal microscopy images indicated that the pyrazine conjugates were selectively taken up by the folate receptor expressing ovarian cancer KB cells.

Both photosensitizer fluorescence photobleaching and singlet oxygen luminescence (SOL) have been measured during ALA-PDT of skin in attempts to estimate PDT dose. However, the relationship of these detected signals to singlet oxygen (¹O₂) dose in a given volume and to its depth distribution are not well understood and difficult to verify experimentally because of the temporal and spatial variations of the essential parameters in PDT. A model for ALA-PDT of normal human skin was developed to simulate the dynamic progress of PDT. The model incorporates Monte Carlo simulations of excitation light fluence and both SOL and PpIX fluorescence signals, ¹O₂-mediated photobleaching mechanism, ground-state oxygen (³O₂) diffusion and perfusion, a cumulative ¹O₂-dependent threshold vascular response and any initial distribution of PpIX. The simulated time-resolved evolution of the instantaneous PpIX fluorescence photobleaching and cumulative SOL signals are examined as functions of irradiance and related to both the time-resolved distribution of cumulative ¹O₂ production at various depths and the average dose in the dermis. The simulations used a green light source at 523 nm. The correlation of SOL signals with the average dose was found to be less irradiance-dependent than that of fluorescence photobleaching, which indicates the great potential of SOL as a clinical dosimetric tool in PDT.

Photodynamic Therapy involves the therapeutic use of photosensitizers in combination with visible light. The subsequent photochemical reactions generate reactive oxygen species which are considered the principal cytotoxic agents to induce cell death. This technique has become widely used in medicine to treat tumors and other nonmalignant diseases. However, there are several factors related to illumination or the photosensitizer that limit an optimal treatment outcome. The use of nanoparticles (NP) for PDT has been proposed as a solution to current shortcomings. In this way, there are NPs that act as carriers for photosensitizers, NPs that absorb the light and transfer the energy to the photosensitizer and NPs that are themselves photodynamically active. In dermatology, the use of topical photosensitizers produces a time dependent inhomogeneous distribution within the tumor, where the stratum corneum is the main barrier to the diffusion of the photosensitizer to the deeper layers of skin. This produces an insufficient photosensitizer accumulation in tumor tissues and therefore, a low therapeutic efficiency in the case of deep lesions. This work focuses in the use of NPs as photosensitizer carriers to improve the actual topical drug distribution in malignant skin tissues. We present a mathematical model of PS distribution in tumor tissue using NPs that takes into account parameters related to nanoparticles binding. Once the concentration profile of NPs into tissue is obtained, we use a photochemical model which allows us to calculate the temporal evolution of reactive oxygen species according to PS distribution calculated previously from NPs profile.

The diagnosis and treatment of pancreaticobiliary malignancy is of major interest to our group. Building on prior work, we undertook a phase I study of verteporfin photodynamic therapy in patients with locally advanced, unresectable, pancreatic cancer. We also initiated an optical diagnostic study using elastic scattering spectroscopy (ESS) of the normal-appearing periampullary duodenal mucosa in vivo to investigate the hypothesis of a field effect in pancreaticobiliary malignancy. In a phase I dose escalation study, patients were treated with interstitial verteporfin PDT via a single fibre, to determine its general safety profile and the optimum treatment parameters needed to achieve effective and safe necrosis of tumour, With increasing light doses, there was a linear increase in the extent of tumour necrosis around the fibre, without serious adverse events. Follow-on studies using multiple fibres are planned. In 30 patients with benign or malignant pancreaticobiliary disease undergoing clinically-indicated endoscopy, ESS spectra were collected from the normal-appearing duodenum and antrum and a diagnostic algorithm generated by principle component and linear discriminant analysis. Pooled data from duodenal sites distal to the ampulla gave a sensitivity of 86% and a specificity of 72% (82% AUC) for the detection of malignancy, whereas those from the periampullary region had a sensitivity of 77% and a specificity of 61% (72% AUC); antral measurements were not able to discriminate with such accuracy. These early results suggest that ESS of the duodenal mucosa could represent a novel minimally invasive diagnostic test for pancreaticobiliary malignancy.

5-Fluorouracil (5-FU) was developed in the 1950s as an anticancer drug and is now widely used to treat many cancers, including colon and breast carcinoma. 5-FU causes fluoronucleotide misincorporation into RNA and DNA, inhibits thymidylate synthase, and leads to growth arrest and apoptosis. For skin precancers (actinic keratoses; AK), 5-FU is prescribed as a topical agent and was essentially the only option for treating widespread AK of the skin prior to FDA approval of photodynamic therapy (PDT) in 1999. PDT is now gradually replacing 5-FU as a preferred treatment for AK, but neither PDT nor 5-FU are effective for true skin cancers (basal or squamous cell), particularly for tumors >1 mm in depth. In our ongoing work to improve the efficacy of PDT for skin cancer, we previously showed that PDT efficacy can be significantly enhanced by preconditioning tumors with methotrexate (MTX), which leads to increased production of protoporphyrin IX (PpIX) in target cells. However, because MTX must be given orally or intravenously, it is considered unacceptable for widespread human use due to potential toxicity. MTX and 5-FU exert similar effects on the thymidylate synthesis pathway, so we reasoned that topical 5-FU could be a potential alternative to MTX. In this paper, exploratory studies that test 5-FU as a preconditioning agent for PDT are presented. In a cutaneous model of squamous cell carcinoma (chemically-induced papillomatous tumors in mice), 5-FU significantly enhances PpIX accumulation and therefore emerges as a new candidate agent for combination therapy with PDT.

Uniform light fluence distribution for patients undergoing photodynamic therapy (PDT) is critical to ensure predictable PDT outcome. However, common practice uses a point source to deliver light to the pleural cavity with the light uniformity monitored by 7 detectors placed within the pleural cavity. To improve the uniformity of light fluence rate distribution, we have used a real-time infrared (IR) tracking camera to track the movement of the light point source. The same tracking device is used to determine the surface contour of the treatment area. This study examines the light fluence (rate) delivered between the measurement and calculation in phantom studies. Isotropic detectors were used for in-vivo light dosimetry. Light fluence rate in the pleural cavity is calculated and compared with the in-vivo calculation. Phantom studies show that the surface contour can be determined with an accuracy of 2 mm, with maximum deviation of 5 mm. We can successfully match the calculated light fluence rates with the in-vivo measurements. Preliminary results indicate that the light fluence rate can have up to 50% deviation compared to the prescription in phantom experiments. The IR camera has been used successfully in pleural PDT patient treatment to track the motion of light source in realtime. We concluded that it is feasible to develop an IR camera based system to guide the motion of the light source to improve the uniformity of light distribution.

We recently developed a novel therapeutic particle, HerGa, for breast cancer treatment and detection. HerGa consists of a tumor-targeted cell penetration protein noncovalently assembled with a gallium-metallated corrole. The corrole is structurally similar to porphyrin, emits intense fluorescence, and has proven highly effective for breast tumor treatment preclinically, without light exposure. Here, we tested HerGa as a photosensitizer for photodynamic therapy and investigated its mechanism of action using multimode optical imaging. Using confocal fluorescence imaging, we observed that HerGa disrupts the mitochondrial membrane potential in situ, and this disruption is substantially augmented by light exposure. In addition, spectral and fluorescence lifetime imaging were utilized to both validate the mitochondrial membrane potential disruption and investigate HerGa internalization, allowing us to optimize the timing for light dosimetry. We observed, using advanced multimode optical imaging, that light at a specific wavelength promotes HerGa cytotoxicity, which is likely to cause disruption of mitochondrial function. Thus, we can identify for the first time the capacity of HerGa as a photosensitizer for photodynamic therapy and reveal its mechanism of action, opening possibilities for therapeutic intervention in human breast cancer management.

The effectiveness of photodynamic treatment depends on several factors including an accurate knowledge of optical properties of the tissue to be treated. In order to correctly determine the needed light dose, the values of tissue optical properties must be well known. In this study we consider how the uncertainties in the measured values of optical properties affect the uncertainty in light dosimetry. By using phantoms with known optical properties we determine the uncertainties for several algorithms currently in the literature. We calculate the light fluence uncertainties using a finite element model solution to the diffusion equation in a cavity geometry. Using these uncertainties we conduct a statistical analysis, building a three dimensional space of uncertainties in μ_a, μ_s', and light dose. By characterizing this space we gain an understanding of the trend in uncertainties and determine which algorithms provide the most accurate light dose.

Photodynamic therapy (PDT) provides an effective option for treatment of tumors and other diseases. In this work, we extended a previous rate-equation model in time domain with consideration of oxygen diffusion in a spherical cell model. Enhanced oxygen diffusion through cell membrane and non-PDT uptake of oxygen inside the cell have been investigated to study their effect on photobleaching and oxidization leading to cell killing within the context of PDT. We found that the improved PDT model can take into the account of the Michaelis-Menten uptake of the oxygen in addition to diffusion which allows quantitative modeling of photobleaching and cell killing.

For the first time, we summarized previous work, representing the mechanism of photodynamic therapy (PDT) and ordering a set of mathematical models. Valid models were devoted to the simulation of PDT process, which involved two major parts, the photodynamic reaction and photothermal effect. The model covered four common lighting modes, which were point light source, collimated light beam, planar light source and linear light source (in vivo optical fiber) of 50mW~400mW various light intensity and energy of 50J~200J. A homogeneous sphere was used as a tissue phantom and different exposure situations were simulated to study the relationship between the light fluence and PDT. The result turned out that low power light with long treatment time was preferred for photodynamic reaction while photothermal effect required high power. In addition, comparison between the results of the continuous light and intermittent light of a point light source was carried out, which validated the experimental results and drew some conclusions of light modulation optimization. Also, by increasing light intensity to the range of 700mW~3W, the photothermal effect of high-intensity light was simulated. The same effect of uniformly distributed gold particles in the tissue was analyzed and discussed.

Biophysical changes such as inflammation and necrosis occur immediately following PDT and may be used to assess the treatment response to PDT treatment in-vivo. This study uses localized reflectance measurements to quantify the scatter changes in tumor tissue occurring in response to verteporfin-based PDT treatment in xenograft pancreas tumors. Nude mice were implanted with subcutaneous AsPC-1 pancreatic tumors cells in matrigel, and allowed to establish solid tumors near 100mm3 volume. The mice were sensitized with 1mg/kg of the active component of verteporfin (benzoporphryin derivative, BPD), one hour before light delivery. The optical irradiation was performed using a 1 cm cylindrical interstitial diffusing tip fiber with 20J of red light (690nm). Tumor tissue was excised progressively and imaged, from 1 day to 4 weeks, after PDT treatment. The tissue sections were stained and analyzed by an expert veterinary pathologist, who provided information on tissue regions of interest. This information was correlated with variations in scattering and absorption parameters elucidated from the spectral images and the degree of necrosis and inflammation involvement was identified. Areas of necrosis and dead cells exhibited the lowest average scatter irradiance signature (3.78 and 4.07 respectively) compared to areas of viable pancreatic tumor cells and areas of inflammation (5.81 and 7.19 respectively). Bilirubin absorbance parameters also showed a lower absorbance value in necrotic tissue and areas of dead cells (0.05 and 0.1 respectively) compared to tissue areas for viable pancreatic tumor cells and areas of inflammation (0.28 and 0.35). These results demonstrate that localized reflectance spectroscopy is an imaging modality that can be used to identify tissue features associated with PDT treatment (e.g. necrosis and inflammation) that can be correlated with histopathologically-reviewed H&E stained slides. Further study of this technique may provide means for automated discrimination of tissue features based on scatter and absorbance maps elucidated from reflectance spectral datasets and provide a valuable tool for treatment response monitoring during PDT and enabling more effective treatment planning. These results are relevant to verteporfin-based PDT trial for treatment pancreatic cancer in non-surgical candidate cases (VERTPAC-1 University College London, PI Pereira), where individualized assessment of damage and response could be beneficial, if this study is proven to be a well-controlled imaging tool.

This is an in vitro study of photodynamic therapy (PDT) in the MG-63 line of human osteosarcoma cells, as mediated by aminolevulinic acid (ALA). The primary goal of this work is to determine the feasibility and effectiveness of treating osteosarcoma through PDT. In addition, this work is aimed at determining whether the resulting cell death occurs through apoptosis or cellular necrosis. The MG-63 cells are treated with increasing concentrations of ALA from 0.1-10 mM ALA, leading to the accumulation of the photosensitizer protoporphyrin IX (PpIX) within the cells. After incubation periods of 4 and 24 hours in ALA, the cells are illuminated by 0-10 J/cm² of 636 nm light in order to activate the PpIX and induce oxidative damage to the cells. Light is administered by an 8x12 array of LED's, which are controlled by an Arduino Duemilanove microcontroller board in order to assure ease of use along with accurate levels of exposure. Controls for this experiment include 0 J/cm² of light exposure for all experimental concentrations of ALA, as well as illuminating cells that have not been incubated in ALA at all experimental levels of illumination. MG-63 cells are analyzed through fluorimetry and MTT assays in order to determine the effectiveness of ALA mediated PDT of osteosarcoma.

In this work we demonstrate an efficiency of transillumination fluorescence imaging for intravital study of photosensitizers pharmacokinetics in tumor-bearing mice. Experiments were performed on CBA mice with subcutaneously transplanted cervical carcinoma. Photosensitizer fotoditazin was used in the therapeutic dose of 10 mg/kg, i.v. Measuring the fluorescence in the whole tumors at different times after injection of photosensitizer, we obtained in vivo data about the drug accumulation and elimination. It was found in vivo that the period of maximum uptake of the photosensitizer in the mouse tumor is from 1 to 8 h post-injection, and its concentration in the tumor tissue at this time is about 2 μg/g. An accumulation of the photosensitizer in the tumor was confirmed by standard methods ex vivo. Noninvasive assessment of pharmacokinetics by transillumination imaging setup provides an opportunity for rapid and cost-effective studies of newly developed photosensitizers.

The effective treatment of metastatic cancer continues to be a challenge due to the highly invasive nature of metastatic lesions and their propensity to develop therapeutic resistance. Optimal therapeutic regimens for peritoneal metastases should have both rapid uptake and penetrate throughout cancerous lesions. Using in vitro models of ovarian cancer, we have found that the cores of tumor nodules are both hypoxic and acidic, rendering most chemotherapeutic agents ineffective, and considerably reducing the therapeutic efficacy of the majority of photodynamic therapy (PDT) and radiation therapy regimens. PDT using EtNBS, a cationic photosensitizer, is a promising approach for treating these hypoxic and acidic nodule cores as it rapidly accumulates into the hypoxic cores of tumor nodules, and is effective even in completely anoxic environments. To improve the uptake rate of EtNBS into cells, we utilized a carboxylic acid terminated derivative of EtNBS that is zwitterionic at biological pH levels. In this study, we investigated the effect of this structural change on PDT efficacy in ovarian cancer cells.

Porphyrins binding and transport to tumor is the one of the central tasks of photodynamic therapy of tumor (PDT). The main carriers of porphyrins (photosensitizers) in the blood are lipoproteins, serum albumin and hemoglobin. In studying the phenomenon of complexation of proteins with ligands must take into considering the real conditions that exist in the organism and, in particular, take into considering the presence of fatty acids in blood. Up to date the role of fatty acids (palmitic and stearic) in the binding of porphyrins with proteins not been determined. A key step in solving of these problems is to determine the binding constants of porphyrin-protein pairs and effect of fatty acids on this process. The most direct and sufficiently accurate methods of solving such problems are complementary methods of absorption and fluorescence spectroscopy. The results of spectral studies on the binding of porphyrins to serum albumin and hemoglobin in the presence of fatty acids demonstrated a significant decrease in the degree of binding pair porphyrin-albumin and porphyrin-hemoglobin with increasing concentrations of fatty acids in solution. The results lead to the conclusion that for hemoglobin the presence in a solution of fatty acids on binding to the porphyrins affected more significantly than for serum albumin. Thus, in natural conditions, when in the blood presented fatty acids the preference between hemoglobin and serum albumin in the binding and in the transport of porphyrins should be given to serum albumin.

PhotoDynamic Therapy (PDT) has been established as a potent and less invasive treatment for different kinds of cancer. Among various attempts to enhance the therapeutics efficacy of PDT, the specific delivery of the PhotoSensitizer (PS) in the tumor is expected to increase its clinical applications, since unwanted accumulation, especially in the skin, impairs the patients' quality of life (prolonged cutaneous photosensitivity). The aim of this study was to engineer Lipid Nanoparticles (LNP) with different sizes and various PS contents, using simple, solvent-free and easily scale up manufacturing processes. Meso-tetra (hydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive compounds for clinical use and it has been successfully applied in the treatment of various indications, such as the head and neck, prostate and pancreatic cancers. Here, a derivative of mTHPC was efficiently incorporated into the lipid core of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. The photophysical and photochemical properties of mTHPC-loaded LNP were studied by measuring absorbance and fluorescence spectra, colloidal stability, particle size and zeta potential, as well as singlet oxygen luminescence. The photocytotoxicity of three selected mTHPC-loaded LNP (25 nm, 45 nm and 95 nm of diameter, respectively) was evaluated on MCF-7 cells, in comparison to free mTHPC, under irradiation at 652 nm with a range of light fluence from 1 to 5 J/cm². All the physico-chemical, photophysical and biological measurements allow us to conclude that LNP is a promising nano-drug delivery system for PDT.

Photodynamic therapy (PDT) for skin cancer is sometimes only partially effective, due to inadequate levels of the fluorescent drug (photosensitizer, PS) and due to heterogeneous distribution of PS within the tissue. To image the PS distribution within skin tumors, we have developed a fluorescence tomography system (FTS) that combines a fluorescence detection array with a high frequency ultrasound (HFUS) transducer. In this paper we describe in vitro and in vivo validation of this new system. The target fluorophore for detection was Protoporphyrin IX (PPIX). Validation experiments were performed in vivo using a subcutaneous tumor model in which A431 tumor-bearing mice were treated with 5-aminolevulinic acid to induce production of PPIX. FTS reconstructions were compared with standard histology and with data from bulk tumor slices imaged ex vivo on a fluorescence scanner. Reconstructed images obtained from the FTS were correlated with the histology and the ex vivo scans, confirming several-fold increases in PPIX fluorescence in the skin and in the tumor relative to the surrounding tissues. Our data demonstrate the feasibility of using the FTS for subsurface imaging of PPIX in skin carcinoma in vivo. Future aims are to use this device for individualized treatment planning, in order to improve overall patient responses to PDT.

Challenges associated with photodynamic therapy (PDT) include the packaging and site-specific delivery of therapeutic agents to the tissue of interest. Nanoscale encapsulation of PDT agents inside targeted virus capsids is a novel concept for packaging and site-specific targeting. The icosahedral MS2 bacteriophage is one potential candidate for such a packaging-system. MS2 has a porous capsid with an exterior diameter of ~28 nm where the pores allow small molecules access to the capsid interior. Furthermore, MS2 presents suitable residues on the exterior capsid for conjugation of targeting ligands. Initial work by the present investigators has successfully demonstrated RNA-based self-packaging of a heterocyclic PDT agent (meso-tetrakis(para-N-trimethylanilinium)porphine, TMAP) into the MS2 capsid. Packaging photoactive compounds in confined spaces could result in energy transfer between the molecules upon photoactivation, which could in turn reduce the production of radical oxygen species (ROS). ROS are key components in photodynamic therapy, and a reduced production could negatively impact the efficacy of PDT treatment. Here, findings are presented from an investigation of ROS generation of TMAP encapsulated within the MS2 capsid compared to free TMAP in solution. Monitoring of ROS production upon photoactivation via a specific singlet oxygen assay revealed the impact on ROS generation between packaged porphyrins as compared to free porphyrin in an aqueous solution. Follow on work will study the ability of MS2-packaged porphyrins to generate ROS in vitro and subsequent cytotoxic effects on cells in culture.

We have developed an empirical method to determine light distribution in optical phantom. This method is based on experimental measurements of light fluence rate as a function of position inside the medium. Milk solution and fat emulsion were chosen as turbid media and a HeNe laser at 633nm was used as light source. Light fluence rate within the phantoms was detected by an isotropic fiber optic probe. The data were collected for a collimated narrow laser beam and arranged in a tridimensional matrix. Using this matrix, simple mathematical operations were used to simulate different conditions of irradiation geometry. Comparison between experimental measurements and mathematical simulations show that our method can be used to recover light distribution in biological tissue for any condition of illumination, since we have previously performed simple measurements in a sample using a narrow beam. Further in vivo studies must be performed to validate the method here proposed.

The objective of this study was to characterize the death mechanism of human epidermoid carcinoma cells (A431) triggered by photodynamic therapy (PDT) with pheophorbide a. First of all, significant inhibition on the survival of A431 cells (< 20 %) was observed when an irradiation dose of 5.1 J/cm² combined with 125 ng/ml of pheophorbide a was applied. Survival rate of human keratinocyte cells was over 70 % under the same PDT parameters, suggesting that pheophorbide a killed cancer cells selectively. Mitochondria were the main target sites where pheophorbide a accumulated. Formation of reactive oxygen species (ROS) was detected after PDT. Addition of antioxidant N-Acetyl cysteine prevented ROS production and increased cell survival thereafter. The decrease in cellular ATP level was also observed at 6 hrs after PDT. Typical apoptotic cellular morphology and a collapse of mitochondrial membrane potential occurred after PDT. The loss of mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosol, followed by activation of caspase-9 and caspase-3. The activation of caspase-3 resulted in poly(ADP-ribose) polymerase (PARP) cleavage in A431 cells, followed by DNA fragmentation. In conclusion, the results demonstrated that pheophorbide a possessed photodynamic action against A431 cells, mainly through apoptosis mediated by mitochondrial intrinsic pathway triggered by ROS.

A photodynamic therapy experiment on 118 inbred white mice with transplanted Ehrlich's tumor (mouse mammary gland adenocarcinoma) is performed to reveal mechanisms of necrosis formation. In 7-10 days the tumor of 1-1.5 cm diameter is formed under skin at the injection point, and PDT procedure is applied. There were used a chlorine type photosensitizer Radachlorine^TM and 662 nm wavelength diode laser. The drug is injected by intravenously at the dose of 40 mg/kg; the irradiation is executed in 2-2.5 hours at the surface dose of about 200 J/cm². Each of the mice had a photochemical reaction in form of destructive changes at the irradiation region with subsequent development of dry coagulation necrosis. After rejection of the necrosis there occurred epithelization of defect tissues in a tumor place. Histological investigations were conducted in different follow-up periods, in 5 and 30 min, 1, 3, 6, and 12 hours, 1, 3, 7 and 28 days after irradiation. They included optical microscopy, immune marker analysis, morphometry with measurements of volume density of epithelium, tumor stroma and necroses, vascular bed. The investigations showed that an important role in damaging mechanisms of photodynamic action belongs to hypoxic injuries of tumor mediated by micro vascular disorders and blood circulatory disturbances. The injuries are formed in a few stages: microcirculation angiospasm causing vessel paresis, irreversible stases in capillaries, diapedetic hemorrhages, thromboses, and thrombovasculitis. It is marked mucoid swelling and fibrinoid necrosis of vascular tissue. Progressive vasculitises result in total vessel obliteration and tumor necrosis.

In photodynamic therapy (PDT), it is essential to accurately determine the light fluence rate distribution from the known treatment geometry and optical properties. The light distribution calculation for intracavitory PDT is a complex problem because the light near the tissue surface inside the cavity is influenced by the geometry of the surrounding tissue, by multiply scattered light inside the cavity, and by possible attenuation of the fluid contained within the cavity. To address this problem we use Monte Carlo simulations as a gold standard to calculate the light fluence in a spherical cavity, both with homogenous and inhomogeneous optical property distributions. The MC code is developed in the Matlab platform with the Fresnel reflection well defined at the tissue-medium boundary, allowing the number of times a photon can be scattered within the cavity to be user specified. We find that increasing the attenuation, causes a decrease the light fluence rate for the same total light power from a point source. The resulting fluence rate is then compared with our empirical model based on diffusion. Preliminary results of comparisons of the MC simulation and the empirical model will be presented. We conclude that the empirical model is adequate for modeling light fluence within the cavity.