This PDF file contains the front matter associated with SPIE Proceedings Volume 10476, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Apoptosis is a pathway to cell death frequently observed after photodynamic therapy (PDT). Sub-cellular photodamage to mitochondria, lysosomes, the ER, or combinations of these targets, can lead to apoptotic death. We have recently investigated another pathway to cell death after PDT termed ‘paraptosis’. This is characterized by extensive cytoplasmic vacuolization, does not involve caspase activation or nuclear fragmentation, requires a brief interval of continued protein synthesis and appears to derive from ER stress. Determinants and further characteristics of PDT-derived paraptosis are explored in the A549 non small-cell lung cancer cell line and in cells derived from head and neck cancer tissues. We provide evidence that ER photodamage and JNK pathway activation are involved in PDT-mediated paraptosis.

Photodynamic therapy (PDT) has traditionally been used as a photochemical surgical tool. However, there are a plethora of cellular molecular events that occur following the process of photoactivation. These cellular molecular nuances following PDT initiation are being increasingly recognized and may be exploited to expand PDT from being applicable in the metastatic setting. Amongst these are microenvironmental factors, selection pressures and the increase or inhibition of cytokine release. Aspects of this broad hypothesis will be discussed in the presentation.

Drug resistance to conventional therapies remains a major cause of treatment failure, tumor recurrence and dismal survival rates for patients with advanced stage cancers. Photodynamic therapy (PDT) provides an opportunity to exploit photochemically-triggered death mechanisms via targeting of sub-cellular, cellular and stromal compartments to overcome treatment resistance in unresponsive populations of stubborn disease. The informed design of mechanism-based combinations is emerging as increasingly important to targeting resistance and improving the efficacy of conventional treatments, while minimizing toxicity. PDT has been shown to synergize with conventional agents and to overcome the evasion pathways that cause resistance. Increasing evidence shows that PDT-based combinations cooperate mechanistically with, and improve the therapeutic index of, traditional chemotherapies. These and other findings emphasize the importance of including PDT as part of comprehensive treatment plans for cancer, particularly in complex disease sites. Identifying effective combinations requires a multi-faceted approach that includes the development of bioengineered cancer models and corresponding image analysis tools. The presentation will focus on the molecular and phenotypic basis of verteporfin PDT-based enhancement of chemotherapeutic efficacy and predictability in complex 3D models and in vivo models, with a particular emphasis on ovarian and pancreatic cancer.

Photodynamic therapy is a promising alternative treatment modality that uses a photosensitizer to kill cancer cells through oxidative damage. However, many tumors contain regions of hypoxia, limiting the overall effectiveness of the technique. Therefore, an image-guided approach to improve tumor oxygenation during photodynamic therapy could result in better, more-reliable outcomes. We have developed nanoparticles that act as ultrasound/photoacoustic imaging contrast agents while also delivering oxygen to these hypoxic tumor sites. The particles contain a perfluorocarbon (PFC) core which, when vaporized, creates an acoustic impedance difference between the particles and surround tissues, allowing the particles to be visualized with ultrasound imaging. In addition to its contrast enhancement, the PFC core is also a great carrier of oxygen, capable of delivering the payload to tumors. Hydrophobically modified indocyanine green dye (IGC) is added as the photosensitizer to absorb the optical energy from the nanosecond pulsed laser that is used to activate the particles and nucleate vaporization. Upon activation of the particles, a bolus of oxygen is released into the surrounding tissue. The release of oxygen can be quantified by imaging the tumor with spectroscopic photoacoustic imaging in real time. Finally, the encapsulated ICG dye can be leveraged to act as a photosensitizer for photodynamic therapy. Experiments show that physiologically relevant payloads of oxygen can be released from the particles on demand. Furthermore, we are able to visualize the vaporized particles with single-particle sensitivity. These results pave the way for improved image-guided photodynamic therapy.

Photodynamic Therapy (PDT) is used clinically for the treatment of Actinic Kerasotis (AK) and non-melanoma skin cancers (NMSC). These treatments utilize the patient’s ability to convert the pro-drug aminolevulinic acid (ALA) to protoporphyrin IX (PpIX) as part of the heme cycle. Conventional PDT treatment requires a period of incubation to increase the concentration of PpIX at the affected site before a high fluence-rate narrow band light is used to activate the PpIX. Upon activation, the release of reactive oxygen species causes localized cell death, but has also been attributed to reports of pain during treatment. Broadband low fluence-rate light sources and shorter incubation times have been shown to reduce the reported pain while still providing similar efficacy. To compare the light dose provided by these various narrowband and broadband sources, a PpIX absorbance weighted light dose is used. Previous work using a murine skin model has shown this metric to be correlated with epidermal damage and STAT3 cross-linking. However, when comparing light sources at different wavelengths it is important to consider the penetration depth of both the PpIX and the light. Monte Carlo simulations have been implemented to better estimate the impact of tissue optical properties and biodistribution of PpIX. These results are compared with in vivo bioassays with the aim of providing a more accurate tool for light-dose estimation in clinical practice.

Introduction: Topical photodynamic therapy (PDT) is a popular treatment for many non-melanoma skin cancers including actinic keratosis, Bowen’s disease, and some basal cell carcinomas. A chief complaint among patients is the pain that sometimes accompanies the procedure. This has led to a surge of interest in continuous, low-fluence rate PDT which is thought to be less painful. There is myriad evidence in the literature to suggest that natural sunlight can be an effective photo-activator of PpIX, the cytotoxic intermediate. However, there is some concern among the dermatological community regarding the degree to which this method of light delivery may be controlled. It is in this vein that we seek to compare the efficacy of natural sunlight with artificial light. Purpose: The present study compares three low-fluence rate light sources in normal mouse skin, combined with ALA-PDT. The light sources of interest are natural sunlight, a 415 nm LED bulb, and a broad spectrum, white light, horticultural bulb. Methods: A PpIX weighted fluence of 20 J/cm^2 was given to each light group, delivered over ~ 2 hours. Acute indicators of PDT efficacy were assayed via PpIX dosimetry, interrogation of Stat3 crosslinking, and analysis of epidermal keratinocytes for morphological changes.

The aim of this study was to compare the effectiveness of Rose Bengal (RB) and Methylene Blue (MB) as photosensitizers (PS) in Photodynamic Inactivation (PDI) on planktonic cultures of Candida albicans, a well-known opportunistic pathogen. RB and MB at concentrations ranging from 0.5 to 60 μM and fluences of 10, 30, 45 and 60 J/cm² were tested. The light sources consist of an array of 12 led diodes with 30 mW of optical power each; 490-540 nm (green light) to activate RB and 600 -650 nm (red light) to activate MB. We first optimize the in vitro PDI technique using a single light dose and the optimum PS concentration. The novelty of our approach consist in reducing further the PS concentration than the optimum obtained with a single light exposure and using smaller light fluence doses by using repetitive light exposures (two to three times). MB and RB were tested for repetitive exposures at concentrations ranging from 0.1 to 10 μM, with fluences of 3 to 20 J/cm², doses well below than those reported previously. All experiments were done in triplicate with the corresponding controls; cells without treatment, light control and dark toxicity control. RB-PDI and MB-PDI significantly reduced the number of CFU/mL when compared to the control groups. The results showed that RB was more effective than MB for C. albicans inactivation. Thus, we show that is possible to reduce significantly the amount of PS and light fluence requirements using repetitive light doses of PDI in vitro.

There are several advantages of Photodynamic Therapy (PDT) for nonmelanoma skin cancer treatment compared to conventional treatment techniques such as surgery, radiotherapy or chemotherapy. Among these advantages its noninvasive nature, the use of non ionizing radiation and its high selectivity can be mentioned. Despite all these advantages, the therapeutic efficiency of the current clinical protocol is not complete in all the patients and depends on the type of pathology. An adequate dosimetry is needed in order to personalize the protocol. There are strategies that try to overcome the current PDT shortcomings, such as the improvement of the photosensitizer accumulation in the target tissue, optical radiation distribution optimization or photochemical reactions maximization. These strategies can be further complemented by the use of nanostructures with conventional PDT.

Customized dosimetry for nanoparticle-based PDT requires models in order to adjust parameters of different nature to get an optimal tumor removal. In this work, a predictive model of nanoparticle-based PDT is proposed and analyzed. Dosimetry in nanoparticle-based PDT is going to be influenced by photosensitizer-nanoparticle distribution in the malignant tissue, its influence in the optical radiation distribution and the subsequent photochemical reactions. Nanoparticles are considered as photosensitizer carriers on several types of non-melanoma skin cancer. Shielding effects are taken into account. The results allow to compare the estimated treatment outcome with and without nanoparticles.

We developed light-triggered liposomes incorporating gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized by adjusting the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of HSPC: PE-NH2: gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of these liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox loaded liposomes were applied to human colorectal cancer cells, HCT116, and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity, compared to the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may have improved therapeutic efficacy in photodynamic therapy and chemotherapy.

Background: The majority of cancers upregulate their transferrin receptor (Tf-R) to satisfy their higher Fe3+ requirements for proliferation. TLD-1433 can bind to transferrin to form Rutherrin, which is a promising photosensitizer with stable chemical structure and higher tissue selectivity. Methods: To investigate the effect of Rutherrin®-mediated photodynamic treatment (PDT), we used non-small lung cancer cell lines H2170, A549, and H460. Subcutaneous tumors were treated with Rutherrin-mediated PDT, 4hrs post intravenous administration. The treatment parameters10 mg/kg Rutherrin and 600 Jcm-2 808 nm radiation. In an orthotopic A549 tumor model, the presence of tumor after inoculation in lungs was confirmed by microCT. Tissue samples were collected for Inductively Coupled Mass Spectrometry to quantify the Rutherrin concentrations via a Ru isotope in tumor and normal lung tissue. Results: Evaluation of TfR expression by flow cytometric and western blotting showed that almost all cancer cells express TfR. In in-vitro cytotoxicity assay, all cancer cell lines showed high cell kill by PDT at 100nM Rutherrin concentrations. In the subcutaneous tumor model, PDT after Rutherrin injection significantly inhibited the tumor growth and histopathology showed extensive necrosis at 24 hrs, which was confirmed with lowered Ki67 staining. In an orthotopic model, the lung lobe with tumor retained more Rutherrin than the contralateral lung, showing specific tumor uptake. Conclusion: These results support the hypothesis that safe and efficient Rutherrin-mediated PDT is feasible due to improved photosensitizer localization to lung tumors tissue. Selective irradiation of the cancer lesions by strategic placement of the light source remains a requirement.

Photodynamic therapy (PDT) is a clinically approved method for the treatment of cancer by using singlet oxygen, a highly reactive oxygen generated from a photosensitizer drug upon photoactivation. Limited light penetration depth into the tissue means that PDT is unsuitable for deep tissue cancer treatments. To overcome this issue, we developed a dual PDT system where poly (D, L-lactide-co-glycolide) (PLGA) polymeric nanoparticles incorporating a photosensitizer, verteporfin, can be triggered to generate cytotoxic singlet oxygen by both red light at 690 nm and by 6 MeV X-ray radiation. The X-ray radiation used in this study allows this system to break through the PDT depth barrier, due to excellent penetration of 6 MeV X-ray radiation though biological tissue. In addition, the conjugation of the nanoparticles with folic acid moieties has enabled specific targeting of HCT116 cancer cells which overexpress the folate receptors. Physiochemical characterization of PLGA nanoparticles, such as size distribution, zeta potential, morphology and in-vitro release of verteporfin was also carried out. These studies indicate that improved tumour cell killing effects can be achieved with nanoparticles triggered by 690 nm as well as 6 MeV radiation, compared with nanoparticles alone. We attribute the X-ray induced singlet oxygen generation from the photosensitizer verteprofin to photoexcitation by Cerenkov radiation and/or chemical reaction facilitated by energetic secondary electrons produced in the tissue. This effect offers the possibility of enhancing the commonly prescribed radiation therapy by simultaneous PDT.

Background: Skin photosensitivity is a major side effect of photodynamic therapy (PDT). It is induced by the photosensitizer remaining in the skin. It is usually rapidly metabolized by the liver, but the pharmacokinetic profile varies widely among individuals. This makes it difficult to predict the incidence of skin photosensitivity. Therefore, we conducted a prospective study to investigate whether the NPe6 fluorescence intensity in skin after NPe6-PDT could be measured safely in human patients using a fluorescence sensing system for judging the risk of skin photosensitivity.
Methods: The NPe6 fluorescence measurements using a constructed fluorescence sensing system at the inside of the arm were acquired prior to and 5 and 10 minutes after NPe6 administration as well as at the time of PDT (4-5 hours after administration), at discharge (2 or 3 days after PDT), and at 1 or 2 weeks after PDT. Participants were interviewed as to whether they had any complications at 2 weeks after PDT.
Results: Nine male patients and one female patient entered this study. All of the measurements of NPe6 fluorescence in the skin could be obtained without any complications. The spectral peak was detected at the time of discharge (2-3 days after administration) in most cases and it decreased at 1 or 2 weeks after PDT.
Conclusions: The fluorescence of NPe6 in the skin could be detected feasibly using the fluorescence sensing system in human patients. Measuring fluorescence intensity in the skin might be useful to predict the incidence of skin photosensitivity after PDT.

In recent years, Phase I/II clinical trials of photodynamic therapy performed at our institute have assessed the safety and potential efficacy of PDT for several indications. Furthermore, these trials have provided a wealth of data on the biology of PDT in clinical application. Clinical findings have confirmed some expectations that stem from preclinical data, but they have also raised new questions based on observations not previously made in animal models. We describe the iterative process used to develop new preclinical models and systems for the purpose of substantiating and then researching novel observations rising from clinical application of PDT. For example, in a clinical trial of PDT for early stage/premalignant head and neck cancer we found clinical data to corroborate preclinical findings that associate tumor oxygenation and PDT outcome. However, these clinical data also identify the potential for physiologically informed light delivery to provide individualized optimization of PDT. Toward this goal, a physiologically informed light delivery system has been developed and is being tested in murine studies. In a second example, clinical trials of PDT for malignant pleural mesothelioma have demonstrated that a stronger inflammatory response to surgical debulking (prior to PDT) associates with shorter overall survival. This observation suggests that mitigation of surgery-induced inflammation may increase the efficacy of intraoperative PDT. Animal models have been developed to study the effect of acute inflammation on PDT, and in ongoing studies the mechanisms of these interactions are being elucidated.

Accurate light dosimery is critical to ensure consistent outcome for pleural photodynamic therapy (pPDT). Ellipsoid shaped cavities with different sizes surrounded by turbid medium are used to simulate the intracavity lung geometry. An isotropic light source is introduced and surrounded by turbid media. Direct measurements of light fluence rate were compared to Monte Carlo simulated values on the surface of the cavities for various optical properties. The primary component of the light was determined by measurements performed in air in the same geometry. The scattered component was found by submerging the air-filled cavity in scattering media (Intralipid) and absorbent media (ink). The light source was located centrally with the azimuthal angle, but placed in two locations (vertically centered and 2 cm below the center) for measurements. Light fluence rate was measured using isotropic detectors placed at various angles on the ellipsoid surface. The measurements and simulations show that the scattered dose is uniform along the surface of the intracavity ellipsoid geometries in turbid media. One can express the light fluence rate empirically as φ =4S/A_s*R_d/(1- R_d), where R_d is the diffuse reflectance, As is the surface area, and S is the source power. The measurements agree with this empirical formula to within an uncertainty of 10% for the range of optical properties studied. GPU voxel-based Monte-Carlo simulation is performed to compare with measured results. This empirical formula can be applied to arbitrary geometries, such as the pleural or intraperitoneal cavity.

Treatment of malignant pleural mesothelioma remains palliative in nature, and consists of surgical resection in order to achieve local control. More recently, surgical procedures which spare the lung (radical pleurectomy) have been coupled with photodynamic therapy (PDT) of residual disease to achieve better local control. Due to increasing evidence of the effects of surgery on both host immunity and residual tumor cells, we investigated the contribution of injuries sustained during surgery to efficacy of photodynamic therapy in a mouse model of malignant mesothelioma. We previously observed that surgical injury prior to PDT inhibits long-term response in vivo. As it relates to PDT outcome, we are now investigating neutrophil profiles in the presence and absence of surgical injury. Our results demonstrate that neutrophil influx into the tumor and lymph node occur sooner when PDT is preceded by surgical injury, as demonstrated by higher neutrophil counts in the respective tissue. Through in vivo imaging using luminol chemiluminescence as a marker of neutrophil activation, we show a role of neutrophil-secreted myeloperoxidase activity in producing long-term response after PDT. However, myeloperoxidase deposition in the lymph node is known to suppress dendritic cell migration, activation, and antigen uptake. Thus, we are currently investigating if early influx and activation of neutrophils in tumor draining lymph nodes results in a loss of establishment of PDT-mediated immunity. Taken together, these studies will describe potential immunomodulatory roles for myeloperoxidase in responses to intraoperative PDT.

Glioblastoma (GBM) has less than one-year survival rate due to local recurrence post treatment or growth of “left-over” residual disease post surgical resection. To personalize treatment strategies and reduce the rate of recurrence, it is important to develop imaging based therapeutic strategies and prediction markers to identify GBM recurrence. Given the role of the vasculature in tumor growth and survival, here we utilize multi-parametric ultrasound and photoacoustic imaging to validate if changes in vascular structure and function could be predictive of treatment response in patient- derived orthotopic Glioblastoma xenograft models. Patient-derived GBM cell lines were implanted in the brain of Swiss nu/nu mice. After the tumors reached 2-4 mm in diameter, the mice were divided into 4 groups namely – no treatment, surgical resection, therapy and surgical resection with therapy. Photodynamic therapy (PDT), a light based cytotoxic therapy, with photosensitizer Benzoporphyrin derivative (BPD) or FDA approved chemotherapy temozolomide were administered in the mice. Fujifilm VisualSonics LAZR system with a 20 MHz transducer was used to obtain power Doppler (%vascularity in tumors) and photoacoustic oxygen saturation (StO2) maps of the brain tumors at different time points pre and post treatment. The mice were either euthanized for immunofluorescence to validate the imaging markers or longitudinally monitored for tumor volume until moribund.The no-treatment group or the surgery only group did not have significant changes in vascular density or StO2. We observed a statistically significant decrease in StO2 immediately post BPD-PDT (primarily a vascular therapy) but not with temozolomide (a cellular therapy). Furthermore, we also observed that the sustenance of hypoxia or low StO2 in tumors for 24-72 hours can be a predictive biomarker for tumor recurrence. Overall, these results suggest the utility of ultrasound and photoacoustic imaging in monitoring treatment response and developing treatment prediction strategies for glioblastoma.

Successful outcome of Photodynamic therapy (PDT) depends on accurate delivery of prescribed light dose. A quality assurance program is necessary to ensure that light dosimetry is correctly measured. We have instituted a QA program that include examination of long term calibration uncertainty of isotropic detectors for light fluence rate, power meter head intercomparison for laser power, stability of the light-emitting diode (LED) light source integrating sphere as a light fluence standard, laser output and calibration of in-vivo reflective fluorescence and absorption spectrometers. We examined the long term calibration uncertainty of isotropic detector sensitivity, defined as fluence rate per voltage. We calibrate the detector using the known calibrated light fluence rate of the LED light source built into an internally baffled 4" integrating sphere. LED light sources were examined using a 1mm diameter isotropic detector calibrated in a collimated beam. Wavelengths varying from 632nm to 690nm were used. The internal LED method gives an overall calibration accuracy of ± 4%. Intercomparison among power meters was performed to determine the consistency of laser power and light fluence rate measured among different power meters. Power and fluence readings were measured and compared among detectors. A comparison of power and fluence reading among several power heads shows long term consistency for power and light fluence rate calibration to within 3% regardless of wavelength. The standard LED light source is used to calibrate the transmission difference between different channels for the diffuse reflective absorption and fluorescence contact probe as well as isotropic detectors used in PDT dose dosimeter.

Over the past several decades hundreds of cancer patients have been treated with PDT in both clinical trials and off label. PDT has been used for a wide variety of malignancies, including lung, esophageal, head and neck and pancreatic cancer, as well as mesothelioma. Treatment regimens and photosensitizer usage has also been variable. The knowledge gained from these studies has helped PDT to move into acceptance within some areas of the medical community, but the progress has been slow. This is due in part to a lack of Phase II and III randomized clinical trials in which PDT is measured against the standard of care. In addition, the wide variety of treatment parameters and study protocols has made it difficult to draw general conclusions on the factors that affect the efficacy of PDT. The knowledge gained from these studies has helped PDT to move into acceptance within some areas of the medical community, but the progress has been slow. The Registry will be grouped by disease site; we have developed lung, esophageal and mesothelioma to date with plans to expand to other sites. Data collected within the registry will include patient characteristics, PDT procedure specifics, outcomes, complications and quality of life assessments. Data will be searchable and the registry will be set up to provide reports to specific inquiries. Registry participation is open to all clinicians and researchers. A demonstration of the registry and its attributes will be given during this talk.

Ocular melanoma is the second most common melanoma type after the cutaneous variety and represents about 3.7% of all melanomas. Of these, 82.5% are uveal, 6.6% are conjunctival and 10.9% occur at other sites. It is a potentially lethal disease, especially when it results in metastatic spread. Conventional treatments include radiation (brachytherapy, teletherapy), transpupillary thermotherapy, surgical resection (partial or full), enucleation, chemotherapy, and immunotherapy, but recurrences are frequent, with high morbidity depending on the initial tumor size. Two-photon photodynamic therapy (2-PE PDT) involves the use of ultrashort (fs) long-wavelength (near-infrared, NIR) laser pulses to excite the photosensitizer rather than low-intensity continuous light. The advantage is that melanin has a low NIR absorption, improving the light penetration into the tumor. Our in vitro studies show that 2-PE PDT is more efficient in melanotic than in amelanotic melanoma cells, while the control 1-PE PDT gives similar responses in both cell lines. In vivo, using conjunctival melanomas grown in 10 mice, histology shows apoptosis only at the treatment site, with no damage to the surrounding normal tissue. Also, in a single session, the technique could eliminate the entire tumor mass. These initial results suggest that 2-PE PDT has a high potential for the treatment of pigmented ocular melanomas and may represent a new alternative to conventional therapies.

Breast cancer (BCA) is the most frequently diagnosed cancer in women, with distant metastases to lung, liver, bone and skin occurring in approximately 40% of cases. Radiation therapy (RT) has been successfully employed for the treatment of BCA; however, multiple rounds of RT are associated with undesirable cutaneous side effects. This study explores PDT as a therapeutic alternative, to be given alone or in combination with RT and chemotherapy. Earlier, we had developed differentiation-enhanced combination photodynamic therapy (cPDT) using a neoadjuvant (5-fluorouracil; 5FU) prior to PDT. The neoadjuvant increases the levels of PpIX, leading to better efficacy following aminolevulinate (ALA)- based PDT. Here, to avoid the toxicity of systemic 5FU, we used a nontoxic 5FU precursor (Capecitabine; CPBN) in a new cPDT regimen. CBPN, a standard chemotherapeutic for BCA, is metabolized to 5FU specifically within tumor tissue. Murine (4T1) BCA cells were injected into breast fat pads of nude mice. CPBN was administered by oral gavage followed by intraperitoneal ALA and red light for PDT. CPBN pretreatment of 4T1 tumors led to increased tumor cell differentiation (3.5 fold), homogenous elevation of intratumoral PpIX levels (4.5 fold), and enhanced tumor cell death post-PDT (5 fold), relative to vehicle control. Using an in vivo imaging system (IVIS), a decline in tumor growth following CPBN-PDT was observed. Results showing the effect of CPBN-PDT on distant metastases of BCA to lung, lymph nodes and skin will be presented. In summary, CPBN-PDT, a novel combination approach, has a significant potential for translation into the clinic.

ALA PDT is a FDA approved cancer treatment. The general model is that excess exogenous ALA is eventually converted to the active photosensitizer, PpIX, and accumulates PpIX to concentrations well above baseline. This accumulation, however, varies considerable from person to person and even intra-tumorally due to a high number of factors that are involved. Due to this there is an increasing desire to pair ALA PDT with other treatments to enhance the efficacy of PDT. This idea itself isn’t new as the labs of Bin Chen and Edward Maytin have a long history of using biology to enhance PpIX accumulation. The PI3K pathway is a long-studied cancer treatment target due to it being one of the most ubiquitous over expressed pathways in cancer and that many treatments have demonstrated enhanced efficacy upon PI3K inhibition. In this paper we show that the PI3K pathway inhibitor, LY294002, alters PpIX accumulation in cells (decreased for A431 and increases for Panc-1 and Panc-1 OR) and significantly increases the efficacy of ALA PDT in every case for both monolayer and spheroid cultures. Additionally, we show that PDT treatments using the nonendogenous photosensitizer, verteporfin, also have enhanced efficacy upon PI3K inhibition. Beyond the treatment synergy of PI3K inhibition and PDT, this work presents a cell pairing model that is perfect to study the previously, to our knowledge, undocumented connection between the PI3K pathway and PpIX accumulation.

Photodynamic therapy (PDT) has been investigated in clinical studies as a treatment method for breast cancer chest wall recurrences. Complete response percentage in these studies is not 100% in most patients, indicating the presence of a remaining tumor after PDT. Some in vitro studies show that tumor cells present distinct threshold dose, suggesting that the remaining tumor in vivo could require higher doses or different PDT strategies. There is still a lot of controversy of the multiple PDT sessions effect on bulky tumors. The purpose of this study is to investigate low-dose PDT parameters in 3D cultures of breast cancer cells grown by the magnetic levitation method. PDT was performed with Photodithazine® (PDZ) and LED irradiation at 660 nm. Two concentrations of PDZ were investigated and the 50 μg/mL concentration, which showed a superficial distribution, was used in the PDT. Partial damage was observed in the tumors and the viability test showed a small percentage of cell death. This outcome is favorable for the investigation of PDT effects in the remaining tumor. Multiple PDT sections could provide more noticeable alterations in cell morphology and metabolism.

Photodynamic therapy (PDT) is a noninvasive phototherapy method that has been clinically approved for many years. During this type of therapy, the photosensitizing agent will be excited by optical photons to generate reactive oxygen species which can kill nearby cancer cells. However, due to the strong optical scattering and absorption of tissue, optical photons can only penetrate tissues in few millimeters which result in the limited applications of PDT to superficial lesions like skin cancers. In this study, to overcome the penetration limitations, we used high-energy photons to excite photosensitizers directly by assuming that high-energy photons generate low-energy optical photons in tissues to excite photosensitizers. Cesium- 137 irradiator has been used as the high-energy photon source. A fiber pigtailed diode laser was used to validate the photosensitizer’s efficacy. We used MPPa as the photosensitizer to treat A549 cancer cell line with different concentrations of drug (10μM/ ml, 5 μM/ml, 2.5 μM/ml, 1 μM/ml and 0 μM/ml). We have performed an irradiation experiment for different time durations of 30 min, 15 min, 7 min to 3 min, respectively, and we also compared different drug concentrations and different exposure durations. Our study not only proved the MPPa PDT method was effective, but also indicated that high-energy photons enhanced PDT could potentially overcome the penetration limitations thus making PDT feasible for deep tissue cancer.

Type I photodynamic therapy (PDT) is based on the use of photochemical reactions mediated through an interaction between a tumor-selective photosensitizer, photoexcitation with a specific wavelength of light, and production of reactive oxygen species (ROS). The goal of this study is to develop a model to calculate reactive oxygen species concentration ([ROS]_rx) after Tookad®-mediated vascular PDT. Mice with radiation-induced fibrosarcoma (RIF) tumors were treated with different light fluence and fluence rate conditions. Explicit measurements of photosensitizer drug concentration were made via diffuse reflective absorption spectrum using a contact probe before and after PDT. Blood flow and tissue oxygen concentration over time were measured during PDT as a mean to validate the photochemical parameters for the ROSED calculation. Cure index was computed from the rate of tumor regrowth after treatment and was compared against three calculated dose metrics: total light fluence, PDT dose, reacted [ROS]_rx. The tumor growth study demonstrates that [ROS]_rx serves as a better dosimetric quantity for predicting treatment outcome, as a clinically relevant tumor growth endpoint.

Photodynamic therapy (PDT) is a technique that combines light’s interaction with a photoactive substance to promote cellular death and that has been used to treat a wide range of maladies. Cancer is among the leading causes of death worldwide and has been a central issue assessed by PDT research and clinical trials over the last 35 years, but its efficiency has been hampered by photosensitizer buildup at treatment site. Nanotechnology has been addressing drug delivery problems by the development of distinct nanostructured platforms capable of increasing pharmacological properties of molecules. The association of nanotechnology’s potential to enhance photosensitizer delivery to target tissues with PDT’s oxidative damage to induce cell death has been rising as a prospect to optimize cancer treatment. In this study, we aim to verify and compare the efficiency of PDT using redox-responsive silica-based nanoparticles carrying protoporphyrin IX (PpIX) in vitro, in both tumor and healthy cells. Dose-response experiments revealed the higher susceptibility of murine melanoma cells (B16-F10 cell line) to PDT (630 nm, 50 J/cm²) when compared to human dermal fibroblasts (HDFn): after 24 h of incubation with 50 μg/mL nanoparticles solutions, approximately 80 % of B16- F10 cells were killed, while similar results were obtained in HDFn cultures when solutions over 150 μg/mL were used. Uptake and ROS generation assays suggest increased nanoparticle internalization in the tumor cell line, in comparison with the healthy cells, and greater ROS levels were observed in B16-F10 cells.

In this study, we used bacteriochlorin as a photosensitizer, characterized by their low toxicity in the absence of light, presenting absorption around 780 nm, with the objective of evaluating their photodynamic inactivation potential on Staphylococcus aureus and Escherichia coli. Bacteriochlorins were synthesized from the extraction of bacteriochlorophylls from non-sulfurous purple bacteria and were then converted to bacteriochlorins. S. aureus and E. coli microorganisms were used in the planktonic and biofilm forms. For the formation of biofilms on glass coverslips, suspensions of the microorganisms at the concentration of 10⁶ CFU/mL were inoculated into each well of a microplate. There was an exchange of culture medium (Tryptic Soy Broth - TSB) every 24 hours for 7 days, pre-washing the coverslips with a phosphate-buffered saline (PBS), to ensure that only adhered microorganisms were grown and then incubated at (36 ± 1)°C between the middle exchanges. After 7 days of induction, the biofilm was mature, like those normally found in nature, and then it was applied different treatments (light doses associated with FS concentrations). At the end of the treatment, the coverslips underwent an ultrasonic disintegration, and the quantitative evaluation of viable cells was performed by plate counting using the plate method in Tryptic Soy Agar (TSA), incubating at (36 ± 1)°C for 24 hours. The results showed that the PDI for E. coli was not successful even when it was more susceptible to the planktonic form, whereas for S. aureus the results showed a reduction in cell viability 6 logs for the planktonic forms, but lower to 1 log in biofilms. Therefore, novel studies using bacteriochlorins and surfactants will be performed to verify the potential of this alternative treatment method.

PDT efficacy depends on the concentration of photosensitizer, oxygen, and light delivery in patient tissues. In this study, we measure the in-vivo distribution of important dosimetric parameters, namely the tissue optical properties (absorption μ_a (λ) and scattering μs ’ (λ) coefficients), photofrin concentration (cphotofrin), blood oxygen saturation (%S_tO₂), and total hemoglobin concentration (THC), before and after PDT. We characterize the inter- and intra-patient heterogeneity of these quantities and explore how these properties change as a result of PDT treatment. The result suggests the need for real-time dosimetry during PDT to optimize the treatment condition depending on the optical and physiological properties.

Drug delivery systems combined multimodal therapy strategies are very promising in cancer theranostic applications. In this work, a new drug-delivery vehicles based on human serum albumin (HSA)-coated gold nanorods (GNR/PSS/HSA NPs) was developed. The success of coating was verified by transmission electron microscopy (TEM), zeta potential and fourier transform infrared spectroscopy (FTIR). Furthermore, it is demonstrated that doxorubicin (DOX) is successfully loaded among multilayered gold nanorods by the electrostatic and hydrophobic force, and DOX@GNR/PSS/HSA NPs were highly biocompatible and stable in various physiological solutions. The NPs possess strong absorbance in nearinfrared (NIR) region, and high photothermal conversion efficiency for outstanding photothermal therapy applications. A bimodal drug release triggered by proteinase or NIR irradiation has been revealed, resulting in a significant chemotherapeutic effect in tumor sites because of the preferential drug accumulation and triggered release. Importantly, the in vitro and in vivo experiments demonstrated that DOX@GNR/PSS/HSA NPs, which combined photothermal and chemotherapy for cancer therapy, revealing a remarkably superior synergistic anticancer effect over either monotherapy. All these results suggested a considerable potential of DOX@GNR/PSS/HSA NPs nano-platform for antitumor therapy.

To study a mechanism of phrenic nerve preservation phenomena during a photosensitization reaction, we investigated an uptake of talaporfin sodium and photosensitization reaction effect on an electric propagation. Right phrenic nerve was completely preserved after superior vena cava isolations using the photosensitization reaction in canine animal experiments, in spite of adjacent myocardium was electrically blocked. We predicted that low drug uptake and/or low photosensitization reaction effect on the nerve might be a mechanism of that phenomena. To investigate uptake to various nerve tissue, a healthy extracted crayfish ventral nerve cord and an extracted porcine phrenic nerve were immersed in 20 μg/ml talaporfin sodium solution for 0-240 min. The mean talaporfin sodium fluorescence brightness increased depending on the immersion time. This brightness saturated around the immersion time of 120 min. We found that talaporfin sodium uptake inside the perineurium which directly related to the electric propagation function was lower than that of outside in the porcine phrenic nerve. To investigate photosensitization reaction effect on electric propagation, the crayfish nerve was immersed into the same solution for 15 min and irradiated by a 663 nm laser light with 120 mW/cm2. Since we found the action potential disappeared when the irradiation time was 25-65 s, we consider that the crayfish nerve does not tolerant to the photosensitization reaction on electric propagation function at atmospheric pressure. From these results, we think that the low uptake of talaporfin sodium inside the perineurium and low oxygen partial pressure of nerve might be the possible mechanism to preserve phrenic nerve in vivo.

We constructed the 3-compartment talaporfin sodium pharmacokinetic model for canine by an optimization using the fluorescence measurement data from canine skin to estimate the concentration in the interstitial space. It is difficult to construct the 3-compartment model consisted of plasma, interstitial space, and cell because there is a lack of the dynamic information. Therefore, we proposed the methodology to construct the 3-compartment model using the measured talaporfin sodium skin fluorescence change considering originated tissue part by a histological observation. In a canine animal experiment, the talaporfin sodium concentration time history in plasma was measured by a spectrophotometer with a prepared calibration curve. The time history of talaporfin sodium Q-band fluorescence on left femoral skin of a beagle dog excited by talaporfin sodium Soret-band of 409 nm was measured in vivo by our previously constructed measurement system. The measured skin fluorescence was classified to its source, that is, specific ratio of plasma, interstitial space, and cell. We represented differential rate equations of the talaporfin sodium concentration in plasma, interstitial space, cell. The specific ratios and a converting constant to obtain absolute value of skin concentration were arranged. Minimizing the squared error of the difference between the measured fluorescence data and calculated concentration by the conjugate gradient method in MATLAB, the rate constants in the 3-compartment model were determined. The accuracy of the fitting operation was confirmed with determination coefficient of 0.98. We could construct the 3-compartment pharmacokinetic model for canine using the measured talaporfin sodium fluorescence change from canine skin.

Resistance to various anthelmintic drugs is reported in many animals and can become a severe problem for human and animal health. In this study, Photogem® and three curcuminoids compounds (curcumin, demethoxycurcumin, bisdemethoxycurcumin) were used as photosensitizers in the photodynamic inactivation (PDI) in the helminth model Caenorhabditis elegans to investigate the ability of this procedure to worm life cycle. Initially, the presence and location of the photosensitizers in the worm's body were verified by fluorescence confocal microscopy. Curcumin was deposited in the digestive tract and Photogem® along the body of the animal in the incubation time of 12 hours with the photosensitizer. Subsequently, a PDI procedure using a LED device was performed to illuminate the worms treated with the photosensitizers. The worms were observed by optical microscopy until 48 hours after the PDI to verify the changes in motility, the presence of eggs and larvae and the number of live worms. Curcuminoids tested separately and in combination and two light doses of 30 J/m² no changes were observed in the life cycle of the worm at concentrations of 2 mM and 1 mM. However, in treatment with Photogem® and a light dose of 100 J/m² a reduction in motility and reproduction of the worm with 0.2 mg/mL was observed after 6 hours of exposure, in addition to the death of most worms at concentrations of 6, 4, and 2 mg/mL. We suggest, therefore, that photodynamic inactivation with Photogem® may present an anthelmintic effect against C. elegans, but there is a need for studies on helminths with parasitic activity.

Both for therapeutic and diagnosis purposes, light dosimetry is generally based on empirical data reported in the literature. It is known that tissue color, hydration and surface roughness influences the light propagation. In this context, it is important to investigate ways to minimize these effects leading to an enhanced phototherapy or photodiagnosis application. This study aims to evaluate how different coupling agents alter the light distribution at the light-phantom interface. Diffuse reflectance measurements were performed in order to compare the light interaction with the phantom with and without the coupling agents.

Phototherapies have been increasingly used in several applications such as the control of pain and inflammatory processes, photodynamic therapy, and even aesthetics uses. After many decades, the dosimetry for those techniques remains challenging. One of the key issues is the lack of homogeneity obtained for tissue illumination, which may limit adequate treatment. Especially concerning lesions, the surface tissue is usually irregular, and the light does not couple to the tissue efficiently to promote an effective treatment. A series of experiments have been performed using optical phantoms, in which coupling was improved by introducing a gel with a low concentration of scattering agents between the fiber and the phantom as an attempt to improve the homogeneity of light distribution within the phantoms. The effects promoted by roughness on phantom tissue surfaces are considerably attenuated when the coupling gel was introduced, resulting in a more uniform illumination pattern that may be used to promote better phototherapy treatments outcome.

Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.

Photodynamic therapy (PDT) has emerged as one promising treatment regimen for several cancer types, with a clinical trial ongoing in pancreatic adenocarcinoma (PDAC). PDT treatment efficacy mainly depends on the combination of light delivery, oxygen availability and photosensitizer uptake, each of which can be limited in pancreas cancer. Therefore, increasing drug uptake in the tumor would make an important impact on treatment outcome. This study was conducted to focus on the issue with drug resistance by examining the relationship between photosensitizer verteporfin and tissue parameters such as collagen and vascular patency. Verteporfin uptake in the tumors was assessed by fluorescence imaging while collagen content and patent vessel area fraction were quantified by evaluating Masson’s Trichrome and Lectin pathology staining images. Two tumor cell lines – AsPC-1 and BxPC-3 – were modeled in nude mice to investigate the impact of different tumor microenvironments. Experimental results highlighted the correlation between vascular patency and verteporfin uptake. Collagen content was found to be an independent factor within each tumor line, but a comparison across two tumor types suggested that collagen area of greater than 10% of tumor cross section reflected a lower verteporfin uptake. It was observed that whole-slice tumor quantifications have showcased some interesting trends which could be greatly enhanced and further supported by regional analysis.