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1.  Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate. 
British Journal of Cancer  1998;77(9):1386-1394.
It has been proposed that the generation of O2 during photodynamic therapy (PDT) may lead to photochemical depletion of ambient tumour oxygen, thus causing acute hypoxia and limiting treatment effectiveness. We have studied the effects of fluence rate on pO2, in the murine RIF tumour during and after PDT using 5 mg kg(-1) Photofrin and fluence rates of 30, 75 or 150 mW cm(-2). Median pO2 before PDT ranged from 2.9 to 5.2 mmHg in three treatment groups. Within the first minute of illumination, median tumour pO2 decreased with all fluence rates to values between 0.7 and 1.1 mmHg. These effects were rapidly and completely reversible if illumination was interrupted. During prolonged illumination (20-50 J cm(-2)) pO2 recovered at the 30 mW cm(-2) fluence rate to a median value of 7.4 mmHg, but remained low at the 150 mW cm(-2) fluence rate (median pO2 1.7 mmHg). Fluence rate effects were not found after PDT, and at both 30 and 150 mW cm(-2) median tumour pO2 fell from control levels to 1.0-1.8 mmHg within 1-3 h after treatment conclusion. PDT with 100 J cm(-2) at 30 mW cm(-2) caused significantly (P = 0.0004) longer median tumour regrowth times than PDT at 150 mW cm(-2), indicating that lower fluence rate can improve PDT response. Vascular perfusion studies uncovered significant fluence rate-dependent differences in the responses of the normal and tumour vasculature. These data establish a direct relationship between tumour pO2, the fluence rate applied during PDT and treatment outcome. The findings are of immediate clinical relevance.
PMCID: PMC2150183  PMID: 9652753
2.  In-vivo singlet oxygen threshold doses for PDT 
Objective
Dosimetry of singlet oxygen (1O2) is of particular interest because it is the major cytotoxic agent causing biological effects for type-II photosensitizers during photodynamic therapy (PDT). An in-vivo model to determine the singlet oxygen threshold dose, [1O2]rx,sh, for PDT was developed.
Material and methods
An in-vivo radiation-induced fibrosarcoma (RIF) tumor mouse model was used to correlate the radius of necrosis to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include five photosensitizer-specific photochemical parameters along with [1O2]rx,sh. Photosensitizer-specific model parameters were determined for benzoporphyrin derivative monoacid ring A (BPD) and compared with two other type-II photosensitizers, Photofrin® and m-tetrahydroxyphenylchlorin (mTHPC) from the literature.
Results
The mean values (standard deviation) of the in-vivo [1O2]rx,sh are approximately 0.56 (0.26) and 0.72 (0.21) mM (or 3.6×107 and 4.6×107 singlet oxygen per cell to reduce the cell survival to 1/e) for Photofrin® and BPD, respectively, assuming that the fraction of generated singlet oxygen that interacts with the cell is 1. While the values for the photochemical parameters (ξ, σ, g, β) used for BPD were preliminary and may need further refinement, there is reasonable confidence for the values of the singlet oxygen threshold doses.
Discussion
In comparison, the [1O2]rx,sh value derived from in-vivo mouse study was reported to be 0.4 mM for mTHPC-PDT. However, the singlet oxygen required per cell is reported to be 9×108 per cell per 1/e fractional kill in an in-vitro mTHPC-PDT study on a rat prostate cancer cell line (MLL cells) and is reported to be 7.9 mM for a multicell in-vitro EMT6/Ro spheroid model for mTHPC-PDT. A theoretical analysis is provided to relate the number of in-vitro singlet oxygen required per cell to reach cell killing of 1/e to in-vivo singlet oxygen threshold dose (in mM). The sensitivity of threshold singlet oxygen dose for our experiment is examined. The possible influence of vascular vs. apoptotic cell killing mechanisms on the singlet oxygen threshold dose is discussed by comparing [1O2]rx,sh for BPD with 3 hr and 15 min drug-light-intervals, with the later being known to have a dominantly vascular effect.
Conclusions
The experimental results of threshold singlet oxygen concentration in an in-vivo RIF tumor model for Photofrin®, BPD, and mTHPC are about 20 times smaller than those observed in vitro. These results are consistent with knowledge that factors other than singlet oxygen-mediated tumor cell killing can contribute to PDT damage in-vivo.
doi:10.1515/plm-2014-0037
PMCID: PMC4410434  PMID: 25927018
photodynamic therapy; singlet oxygen production; oxygen dependence of singlet oxygen quantum yield
3.  Increasing Damage to Tumor Blood Vessels during Motexafin Lutetium-PDT through Use of Low Fluence Rate 
Radiation research  2010;174(3):331-340.
Photodynamic therapy (PDT) with low light fluence rate has rarely been studied in protocols that use short drug–light intervals and thus deliver illumination while plasma concentrations of photosensitizer are high, creating a prominent vascular response. In this study, the effects of light fluence rate on PDT response were investigated using motexafin lutetium (10 mg/kg) in combination with 730 nm light and a 180-min drug–light interval. At 180 min, the plasma level of photosensitizer was 5.7 ng/μl compared to 3.1 ng/mg in RIF tumor, and PDT-mediated vascular effects were confirmed by a spasmodic decrease in blood flow during illumination. Light delivery at 25 mW/cm2 significantly improved long-term tumor responses over that at 75 mW/cm2. This effect could not be attributed to oxygen conservation at low fluence rate, because 25 mW/cm2 PDT provided little benefit to tumor hemoglobin oxygen saturation. However, 25 mW/cm2 PDT did prolong the duration of ischemic insult during illumination and was correspondingly associated with greater decreases in perfusion immediately after PDT, followed by smaller increases in total hemoglobin concentration in the hours after PDT. Increases in blood volume suggest blood pooling from suboptimal vascular damage; thus the smaller increases after 25 mW/cm2 PDT provide evidence of more widespread vascular damage, which was accompanied by greater decreases in clonogenic survival. Further study of low fluence rate as a means to improve responses to PDT under conditions designed to predominantly damage vasculature is warranted.
doi:10.1667/RR2075.1
PMCID: PMC2995951  PMID: 20726728
4.  Factors Influencing Tumor Response to Photodynamic Therapy Sensitized by Intratumor Administration of Methylene Blue 
Lasers in surgery and medicine  2010;42(8):728-735.
Background and Objective
We examined tumor response to methylene blue (MB)-mediated photodynamic therapy (PDT) in a murine tumor model. The goal was to investigate the effects of drug-light interval (DLI), injection vehicle, and fluence on tumor destruction. Fluorescence and reflectance spectroscopy informed our understanding.
Materials and Methods
EMT6 tumor cells were implanted intradermally on the backs of female BALB/c mice and grown to ~ 4-mm diameter. Mice were given a 35 μL, single site, intratumor injection of 500 μg/mL MB administered in either a water or a 5% ethanol-5% Cremophor-90% saline vehicle. PDT was begun either immediately or after a 1-hour DLI with a fluence rate of 60 mW/cm2. Each animal received a fluence of 240 or 480 J/cm2. Fluorescence and reflectance spectra were captured before and during irradiation.
Results
A protocol consisting of the Cremophor-based vehicle, 0 DLI, and a fluence of 480 J/cm2 was the most effective, with a 55% cure rate as measured by no evidence of tumor 90 days after PDT. Use of the water vehicle with this fluence and DLI reduced the cure rate to 20%. Reducing the fluence to 240 J/cm2 similarly reduced treatment efficacy with 0 and 1-h DLIs. Univariate Cox proportional hazards analysis identified increased fluence, 0 vs. 1-h DLI, and the Cremophor vs. water vehicle as highly significant independent predictors of long term tumor control (p < 0.01 in each case). Multivariate analysis with model selection revealed fluence and injection vehicle as the best predictors of survival hazards. Fluorescence spectroscopy in vivo showed that MB fluorescence decreased monotonically during a 2-h dark interval but was restored by irradiation. Reflectance spectroscopy revealed that MB at this injected concentration attenuates the treatment beam significantly.
Conclusion
Sensitizer delivery vehicle, drug-light interval, and fluence contribute significantly to the tumor response to MB-mediated PDT.
doi:10.1002/lsm.20962
PMCID: PMC2948599  PMID: 20848552
fluorescence spectroscopy; methylene blue; photodynamic therapy; reflectance spectroscopy
5.  In-vivo outcome study of HPPH mediated PDT using singlet oxygen explicit dosimetry (SOED) 
Type II 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 singlet oxygen. However, the medical application of this technique has been limited due to inaccurate PDT dosimetric methods. The goal of this study is to examine the relationship between outcome (in terms of tumor growth rate) and calculated reacted singlet oxygen concentration ([1O2]rx) after HPPH-mediated PDT to compare with other PDT dose metrics, such as PDT dose or total light fluence. Mice with radiation-induced fibrosarcoma (RIF) tumors were treated with different light fluence and fluence rate conditions. Explicit measurements of photosensitizer drug concentration and tissue optical properties via fluorescence and absorption measurement with a contact probe before and after PDT were taken to then quantify total light fluence, PDT dose, and [1O2]rx based on a macroscopic model of singlet oxygen. In addition, photobleaching of photosenitizer were measured during PDT as a second check of the model. Changes in tumor volume were tracked following treatment and compared to the three calculated dose metrics. The correlations between total light fluence, PDT dose, reacted [1O2]rx and tumor growth demonstrate that [1O2]rx serves as a better dosimetric quantity for predicting treatment outcome and a clinically relevant tumor growth endpoint.
doi:10.1117/12.2076441
PMCID: PMC4437638  PMID: 25999656
photodynamic therapy; singlet oxygen; HPPH photosensitizer; in-vivo mice study
6.  Comparison of singlet oxygen threshold dose for PDT 
Macroscopic modeling of singlet oxygen (1O2) is of particular interest because it is the major cytotoxic agent causing biological effects for type II photosensitizers during PDT. We have developed a macroscopic model to calculate reacted singlet oxygen concentration ([1O2]rx for PDT. An in-vivo RIF tumor mouse model is used to correlate the necrosis depth to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include 4 photosensitizer specific photochemical parameters along with the apparent singlet oxygen threshold concentration. Photosensitizer specific model parameters are determined for several type II photosensitizers (Photofrin, BPD, and HPPH). The singlet oxygen threshold concentration is approximately 0.41 – 0.56 mM for all three photosensitizers studied, assuming that the fraction of singlet oxygen generated that interacts with the cell is (f = 1). In comparison, value derived from other in-vivo mice studies is 0.4 mM for mTHPC. However, the singlet oxygen threshold doses were reported to be 7.9 and 12.1 mM for a multicell in-vitro EMT6/Ro spheroid model for mTHPC and Photofrin PDT, respectively. The sensitivity of threshold singlet oxygen dose for our experiment is examined. The possible influence of vascular vs. apoptotic cell killing mechanism on the singlet oxygen threshold dose is discussed using the BPD with different drug-light intervals 3 hrs vs. 15 min. The observed discrepancies between different experiments warrant further investigation to explain the cause of the difference.
doi:10.1117/12.2039719
PMCID: PMC4437725  PMID: 25999651
photodynamic therapy; singlet oxygen production; oxygen dependence of singlet oxygen quantum yield
7.  CATIONIC CERAMIDES AND ANALOGUES, LCL30 AND LCL85, AS ADJUVANTS TO PHOTODYNAMIC THERAPY OF TUMORS 
Photodynamic therapy (PDT) is known to alter the expression of various genes in treated cells. This prompted us to examine the activity of genes encoding two important enzymes in sphingolipid (SL) metabolism, dihydroceramide desaturase (DES) and sphingosine kinase (SPHK), in mouse SCCVII tumor cells treated by PDT using either the porphyrin-based photosensitizer Photofrin or silicon phthalocyanine Pc4. The results revealed that PDT induced an upregulation in the expression of two major isoforms of both genes (DES1 and DES2 as well as SPHK1 and SPHK2). While the changes were generally moderate (2-3 fold gains), the increase in DES2 expression was more pronounced and it was much greater with Photofrin-PDT than with Pc4-PDT (over 23-fold vs. less than 5-fold). Combining either Photofrin-PDT or Pc4-PDT with the cationic C16-ceramide LCL30 (20 mg/kg i.p.) for treatment of subcutaneously growing SCCVII tumors rendered important differences in the therapy outcome. Photofrin-PDT, used at a dose that attained good initial response but no tumor cures, produced 50% cures when combined with a single LCL30 treatment. In contrast, the same LCL30 treatment combined with Pc4-PDT had no significant effect on tumor response. The optimal timing of LCL30 injection was immediately after Photofrin-PDT. The therapeutic benefit was lost when LCL30 was given in two 20 mg/kg injections encompassing intervals before and after PDT. LCL85, the cationic B13 ceramide analogue and SL-modulating agent, also increased cure rates of Photofrin-PDT treated tumors, but the therapeutic benefit was less pronounced than with LCL30. These results with LCL30 and LCL85, and our previous findings for LCL29 (another SL analogue), assert the potential of SLs for use as adjuvants to augment the efficacy of PDT-mediated tumor destruction.
doi:10.1016/j.jphotobiol.2013.06.013
PMCID: PMC3770264  PMID: 23911762
Sphingolipid analogs; Squamous cell carcinoma SCCVII; Photodynamic therapy; Dihydroceramide desaturase; Sphingosine kinase
8.  In-vivo Light dosimetry for pleural PDT 
Proceedings of SPIE  2009;7164:71640A.
In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is one of the critical dosimetry quantities for predicting PDT outcome. This study examines the relationship between the PDT treatment time and thoracic treatment volume and surface area for patients undergoing pleural PDT. In addition, the mean light fluence (rate) and its accuracy were quantified. The patients studied here were enrolled in Phase II clinical trial of Photofrin-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. The ages of the patients studied varied from 34 to 69 years old. All patients were administered 2mg per kg body weight Photoprin 24 hours before the surgery. Patients undergoing photodynamic therapy (PDT) are treated with laser light with a light fluence of 60 J/cm2 at 630nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) was monitored at 7 different sites during the entire light treatment delivery. Isotropic detectors were used for in-vivo light dosimetry. The anisotropy of each isotropic detector was found to be within 30%. The mean fluence rate deliver varied from 37.84 to 94.05 mW/cm2 and treatment time varied from 1762 to 5232s. We found a linear correlation between the total treatment time and the treatment area: t (sec) = 4.80 A (cm2). A similar correlation exists between the treatment time and the treatment volume: t (sec) = 2.33 V (cm3). The results can be explained using an integrating sphere theory and the measured tissue optical properties assuming that the saline liquid has a mean absorption coefficient of 0.05 cm−1. Our long term accuracy studies confirmed light fluence rate measurement accuracy of ±10%. The results can be used as a clinical guideline for future pleural PDT treatment.
doi:10.1117/12.809548
PMCID: PMC4409008  PMID: 25914792
Light dosimetry; photodynamic therapy; Photofrin
9.  Disruption of the Blood–Brain Barrier Following ALA-Mediated Photodynamic Therapy 
Lasers in surgery and medicine  2008;40(8):535-542.
Background and Objective
Photodynamic therapy (PDT) is a local antineoplastic treatment with the potential for tumor cell specificity. PDT using either hematoporphyrin derivatives or 5-aminolevulinic acid (ALA) has been reported to induce brain edema indicating disruption of the blood–brain barrier (BBB). We have evaluated the ability of ALA-mediated PDT to open the BBB in rats. This will permit access of chemotherapeutic agents to brain tumor cells remaining in the resection cavity wall, but limit their penetration into normal brain remote from the site of illumination.
Study Design/Materials and Methods
ALA-PDT was performed on non-tumor bearing inbred Fischer rats at increasing fluence levels. Contrast T1-weighted high field (3 T) magnetic resonance imaging (MRI) scans were used to monitor the degree of BBB disruption which could be inferred from the intensity and volume of the contrast agent visualized.
Results
PDT at increasing fluence levels between 9 and 26 J demonstrated an increasing contrast flow rate. A similar increased contrast volume was observed with increasing fluence rates. The BBB was found to be disrupted 2 hours following PDT and 80–100% restored 72 hours later at the lowest fluence level. No effect on the BBB was observed if 26 J of light was given in the absence of ALA.
Conclusion
ALA-PDT was highly effective in opening the BBB in a localized region of the brain. The degradation of the BBB was temporary in nature at fluence levels of 9 J, opening rapidly following treatment and significantly restored during the next 72 hours. No signs of tissue damage were seen on histological sections at this fluence level. However, higher fluences did demonstrate permanent tissue changes localized in the immediate vicinity of the light source.
doi:10.1002/lsm.20670
PMCID: PMC2667623  PMID: 18798293
brain edema; fischer rat; fluence; fluence rate; magnetic resonance imaging; malignant glioma
10.  The Effects of Ultra Low Fluence Rate Single and Repetitive Photodynamic Therapy on Glioma Spheroids 
Lasers in surgery and medicine  2009;41(8):578-584.
Background and Objective
Achieving local control of gliomas with photodynamic therapy (PDT) requires the delivery of adequate light fluences to depths of 1–2 cm in the resection margin where the majority of local recurrences originate. This is clinically impractical with current single-shot, intraoperative PDT treatments due to the length of time required to deliver adequate fluences. Multiple or extended treatment protocols would therefore seem to be required. The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated PDT using single or, repetitive light delivery protocols was investigated at both low and ultra low fluence rates.
Study Design/Materials and Methods
Human glioma spheroids (400 μm diameter) were subjected to sub-threshold light fluence (1.5, 3, or 6 J cm−2) ALA–PDT consisting of four light delivery schemes: single treatment given over either 1 or 24 hours, repetitive treatment given either as four 1 hour light treatments separated by a 4 day interval, or 24 hours light delivery, consisting of four 24 hours treatments separated by a 3 day interval. Treatment efficacy was evaluated using a growth assay. In some cases, confocal microscopy was used to image cell viability.
Results
The repetitive and single light treatment protocols were most effective when delivered at ultra low (μW cm−2) fluence rates. In all cases, growth inhibition was light dose-dependent. The repetitive ultra low fluence rate treatment (1.5 J cm−2; irradiance = 17 μW cm−2) light delivery protocol was the most effective resulting in total growth inhibition during the 2-week observation period.
Conclusion
Ultra low light fluence rate ALA–PDT results in significant spheroid growth inhibition. Repeated administration of ALA was required during repetitive and/or protracted single PDT treatment protocols. The existence of a lower fluence rate limit, below which the efficacy of threshold light fluences diminish was not found in these studies.
doi:10.1002/lsm.20808
PMCID: PMC4153363  PMID: 19731298
photodynamic therapy; 5-aminolevulinic acid; human glioma spheroids; fluence; fluence rate; repetitive PDT; chronic PDT; malignant glioma
11.  Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters 
Theranostics  2012;2(9):827-839.
Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.
doi:10.7150/thno.4334
PMCID: PMC3475211  PMID: 23082096
Photodynamic therapy; PDT; photosensitizer imaging; fractionation; verteporfin; BPD; in vitro 3D tumor model.
12.  Photodynamic Therapy Using Intra-Articular Photofrin for Murine MRSA Arthritis: Biphasic Light Dose Response for Neutrophil-Mediated Antibacterial Effect 
Lasers in surgery and medicine  2011;43(3):221-229.
Background and Objective
Bacterial arthritis does not respond well to antibiotics and moreover multidrug resistance is spreading. We previously tested photodynamic therapy (PDT) mediated by systemic Photofrin® in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) arthritis, but found that neutrophils were killed by PDT and therefore the infection was potentiated.
Study Design/Materials and Methods
The present study used an intra-articular injection of Photofrin® and optimized the light dosimetry in order to maximize bacterial killing and minimize killing of host neutrophils. MRSA (5 × 107 CFU) was injected into the mouse knee followed 3 days later by 1 μg of Photofrin® and 635-nm diode laser illumination with a range of fluences within 5 minutes. Synovial fluid was sampled 6 hours or 1–3, 5, and 7 days after PDT to determine MRSA colony-forming units (CFU), neutrophil numbers, and levels of cytokines.
Results
A biphasic light dose response was observed with the greatest reduction of MRSA CFU seen with a fluence of 20 J cm−2, whereas lower antibacterial efficacy was observed with fluences that were either lower or higher. Consistent with these results, a significantly higher concentration of macrophage inflammatory protein-2, a CXC chemokine, and greater accumulation of neutrophils were seen in the infected knee joint after PDT with a fluence of 20 J cm−2 compared to fluences of 5 or 70 J cm−2.
Conclusion
PDT for murine MRSA arthritis requires appropriate light dosimetry to simultaneously maximize bacterial killing and neutrophil accumulation into the infected site, while too little light does not kill sufficient bacteria and too much light kills neutrophils and damages host tissue as well as bacteria and allows bacteria to grow unimpeded by host defense.
doi:10.1002/lsm.21037
PMCID: PMC3071702  PMID: 21412806
photoinactivation; antimicrobial effect; neutrophil-mediated host defense; chemokine; macrophage inflammatory protein-2
13.  Enhancement of photodynamic therapy by mitomycin C: a preclinical and clinical study. 
British Journal of Cancer  1996;73(8):945-951.
Photodynamic therapy (PDT) using Photofrin was used in combination with a hypoxic toxin (mitomycin C, MMC) to treat four patients with recurrent skin metastasis of a mammary carcinoma. In preclinical experiments an additive effect was found for the combination of MMC and PDT for treating subcutaneous RIF1 tumours in mice. When interstitial PDT was combined with a low dose of MMC (administered 15 min before illumination), the Photofrin dose or light dose could be reduced by a factor of 2 in order to obtain equivalent cure rate or growth delay. In the clinical pilot study, a low dose of Photofrin (0.75 mg kg-1) was used for PDT alone (superficial illumination) or combined with low-dose MMC (5 mg m-2). Different tumour areas were illuminated with or without a preceding infusion of MMC. Both tumour response and skin photosensitivity were scored. After 8-12 weeks of treatment, tumour cure could be achieved by administering light doses > or = 150 J cm-2 for PDT alone and similar effects were obtained when light doses of 75-87.5 J cm-2 were given after infusion with MMC. In all cases necrotic tissue of both tumour and surrounding skin was observed, which lasted for a mean of 5 months (range 2-20 months). Skin phototoxicity, tested by using a standardised illumination of skin patches on the back, lasted maximally 3 weeks. Three main conclusions could be drawn from these studies: (1) The enhanced effects of the combination of PDT and MMC observed in mouse tumours can be extrapolated to patients with mammary skin metastasis. (2) The combination of PDT and hypoxic toxins facilitates treatment by permitting lower doses of photosensitiser to be used (thereby reducing skin phototoxicity) or lower light doses (thereby reducing illumination times and allowing the possibility to treat larger tumour areas). (3) Restoration of skin after PDT in previously treated tumour areas (chemotherapy, radiation therapy and surgery) is very low.
Images
PMCID: PMC2075831  PMID: 8611430
14.  Tumor Vascular Microenvironment Determines Responsiveness to Photodynamic Therapy 
Cancer Research  2012;72(8):2079-2088.
The efficacy of photodynamic therapy (PDT) depends upon the delivery of both photosensitizing drug and oxygen. In this study, we hypothesized that local vascular microenvironment is a determinant of tumor response to PDT. Tumor vascularization and its basement membrane (collagen) were studied as a function of supplementation with basement membrane matrix (Matrigel) at the time of tumor cell inoculation. Effects on vascular composition with consequences to tumor hypoxia, photosensitizer uptake and PDT response were measured. Matrigel-supplemented tumors developed more normalized vasculature, composed of smaller and more uniformly-spaced blood vessels than their unsupplemented counterparts, but these changes did not affect tumor oxygenation or PDT-mediated direct cytotoxicity. However, PDT-induced vascular damage increased in Matrigel-supplemented tumors, following an affinity of the photosensitizer Photofrin for collagen-containing vascular basement membrane coupled with increased collagen content in these tumors. The more highly-collagenated tumors demonstrated more vascular congestion and ischemia after PDT, along with a higher probability of curative outcome that was collagen dependent. In the presence of photosensitizer-collagen localization, PDT effects on collagen were evidenced by a decrease in its association with vessels. Together, our findings demonstrate that photosensitizer localization to collagen increases vascular damage and improves treatment efficacy in tumors with greater collagen content. The vascular basement membrane is thus identified to be a determinant of therapeutic outcome in PDT of tumors.
doi:10.1158/0008-5472.CAN-11-3744
PMCID: PMC3328659  PMID: 22374982
collagen; photodynamic therapy; microenvironment; normalization; vasculature
15.  Fluence Rate-Dependent Photobleaching of Intratumorally-Administered Pc 4 Does Not Predict Tumor Growth Delay 
Photochemistry and photobiology  2012;88(5):1273-1279.
We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg/kg Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW/cm2 to a fluence of 100 J/cm2. While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared to drug-only controls. There was no significant difference in tumor responses to these two irradiances (p = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW/cm2 group, enhanced photobleaching was associated with prolonged growth delay (p = 0.188), while at 150 mW/cm2 this trend was reversed (p = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc4-PDT under these treatment conditions.
doi:10.1111/j.1751-1097.2012.01171.x
PMCID: PMC3431450  PMID: 22582826
16.  Explicit dosimetry for photodynamic therapy: macroscopic singlet oxygen modeling 
Journal of biophotonics  2010;3(5-6):304-318.
Singlet oxygen (1O2) is the major cytotoxic agent responsible for cell killing for type-II photodynamic therapy (PDT). An empirical four-parameter macroscopic model is proposed to calculate the “apparent reacted 1O2 concentration”, [1O2]rx, as a clinical PDT dosimetry quantity. This model incorporates light diffusion equation and a set of PDT kinetics equations, which can be applied in any clinical treatment geometry. We demonstrate that by introducing a fitting quantity “apparent singlet oxygen threshold concentration” [1O2]rx,sd, it is feasible to determine the model parameters by fitting the computed [1O2]rx to the Photofrin-mediated PDT-induced necrotic distance using interstitially-measured Photofrin concentration and optical properties within each mouse. After determining the model parameters and the [1O2]rx,sd, we expect to use this model as an explicit dosimetry to assess PDT treatment outcome for a specific photosensitizer in an in vivo environment. The results also provide evidence that the [1O2]rx, because it takes into account the oxygen consumption (or light fluence rate) effect, can be a better predictor of PDT outcome than the PDT dose defined as the energy absorbed by the photosensitizer, which is proportional to the product of photosensitizer concentration and light fluence.
doi:10.1002/jbio.200900101
PMCID: PMC3071971  PMID: 20222102
photodynamic therapy; explicit dosimetry; singlet oxygen; mathematical modeling
17.  A Treatment Planning System for Pleural 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. To improve the uniformity of light fluence rate distribution, we have developed a treatment planning system using an infrared camera to track the movement of the point source. This study examines the light fluence (rate) delivered to chest phantom to simulate a patient undergoing pleural PDT. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) was monitored at 7 different sites during the entire light treatment delivery. Isotropic detectors were used for in-vivo light dosimetry. Light fluence rate in the pleural cavity is also calculated using the diffusion approximation with a finite-element model. We have established a correlation between the light fluence rate distribution and the light fluence rate measured on the selected points based on a spherical cavity model. Integrating sphere theory is used to aid the calculation of light fluence rate on the surface of the sphere as well as inside tissue assuming uniform optical properties. The resulting treatment planning tool can be valuable as a clinical guideline for future pleural PDT treatment.
doi:10.1117/12.843044
PMCID: PMC4438758  PMID: 26005242
Photodynamic therapy; light fluence; light dosimetry; intracavitary treatment planning
18.  Efficacy of 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a in photodynamic therapy of human esophageal squamous cancer cells 
Oncology Letters  2013;6(4):1111-1119.
The present study investigated the effects of 2-(1-hexyloxyethyl)-2-devinylpyro pheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) on in vitro cell survival and in vivo tumor growth derived from human esophageal squamous cancer cells (Eca109). A cell counting kit 8 (CCK8) assay was used to assess the phototoxicity of HPPH-mediated PDT in cultured Eca109 cells. The inhibition of tumor growth was determined by the changes in the relative tumor volume (RTV) and tumor weight. The results revealed that HPPH, in the range of 0.005–1 μg/ml, exhibited no cytotoxicity in the Eca109 cells without light exposure and that the in vitro efficiency of HPPH-mediated PDT was higher compared with that of Photofrin®-mediated PDT. The in vivo results indicated that graded doses of HPPH-mediated PDT significantly inhibited the xenograft tumor growth derived from the Eca109 cells in a dose-dependent manner. The inhibition efficacy of 0.6 and 1.0 mg/kg HPPH-mediated PDT was similar to that of 10 mg/kg Photofrin-mediated PDT. Furthermore, HPPH possessed a lower toxicity than Photofrin at the dose that achieved the same efficacy in mice bearing Eca109 subcutaneous tumors. The histopathological findings indicated that the tumor tissues in the photosensitizer (PS)-treated mice demonstrated varying degrees of necrosis. HPPH and Photofrin exhibited vascular cytotoxicity on the treated tumors. In conclusion, the present study demonstrated that the phototoxicity of HPPH-mediated PDT is higher than that of Photofrin-mediated PDT of the same dose. HPPH possessed lower toxicity than Photofrin at the dose that achieved the same efficacy. Therefore, HPPH may be a promising agent for treating human esophageal squamous cell cancer (ESCC).
doi:10.3892/ol.2013.1493
PMCID: PMC3796395  PMID: 24137473
2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a photodynamic therapy; human esophageal squamous cell cancer
19.  Strong and prolonged induction of c-jun and c-fos proto-oncogenes by photodynamic therapy. 
British Journal of Cancer  1996;74(1):30-36.
Photodynamic therapy (PDT) is currently under investigation in phase II and III clinical studies for the treatment of tumours in superficial localisations. Thus far, the underlying mechanisms of PDT regarding cellular responses and gene regulation are poorly understood. Photochemically generated singlet oxygen (1O2) is mainly responsible for cytotoxicity induced by PDT. If targeted cells are not disintegrated, photo-oxidative stress leads to transcription and translation of various stress response and cytokine genes. Tumour necrosis factor (TNF) alpha, interleukin (IL) 1 and IL-6 are strongly induced by photodynamic treatment, supporting inflammatory action and immunological anti-tumour responses. To investigate the first steps of gene activation, this study focused on the proto-oncogenes c-jun and c-fos, both coding for the transcription factor activator protein 1 (AP-1), which was found to mediate IL-6 gene expression. We here determine the effects of photodynamic treatment on transcriptional regulation and DNA binding of transcription factor AP-1 in order to understand the modulation of subsequent regulatory steps. Photodynamic treatment of epithelial HeLa cells was performed by incubation with Photofrin and illumination with 630 nm laser light in vitro. Expression of the c-jun and c-fos genes was determined by way of Northern blot analysis, and DNA-binding activity of the transcription factor AP-1 was evaluated by electrophoretic mobility shift assay (EMSA). Photofrin-mediated photosensitisation of HeLa cells resulted in a rapid and dose-dependent induction of both genes but preferential expression of c-jun. Compared with the transient expression of c-jun and c-fos by phorbol ester stimulation, photodynamic treatment led to a prolonged activation pattern of both immediate early genes. Furthermore, mRNA stability studies revealed an increased half-life of c-jun and c-fos transcripts resulting from photosensitisation. Although mRNA accumulation after PDT was stronger and more prolonged compared with phorbol ester stimulation, with regard to AP-1 DNA-binding activity, phorbol ester was more efficient. Surprisingly, in addition to the activation of AP-1 DNA-binding via PDT, photodynamic treatment can decrease AP-1 DNA-binding of other strong inducers, such as the protein kinase C-mediated pathway of phorbol esters and the antioxidant pyrrolidine dithiocarbamate (PDTC). This study demonstrates a strong induction of c-jun and c-fos expression by PDT, with prolonged kinetics and mRNA stabilisation as compared with activation by phorbol esters. Interestingly, this observation is not coincident with an overinduction of AP-1 DNA-binding, hence suggesting that post-translational modifications are dominant regulatory mechanisms after PDT that tightly control AP-1 activity in the nucleus thus limiting the risk of deregulated oncogene expression.
Images
PMCID: PMC2074600  PMID: 8679454
20.  Light dosimetry and dose verification for pleural PDT 
In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is critical for predicting PDT outcome. Patients in this study are enrolled in a Phase I clinical trial of HPPH-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. They are administered 4mg per kg body weight HPPH 48 hours before the surgery and receive light therapy with a fluence of 15–45 J/cm2 at 661 and 665nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) are monitored at 7 sites during the light treatment delivery using isotropic detectors. Light fluence (rate) delivered to patients is examined as a function of treatment time, volume and surface area. In a previous study, a correlation between the treatment time and the treatment volume and surface area was established. However, we did not include the direct light and the effect of the shape of the pleural surface on the scattered light. A real-time infrared (IR) navigation system was used to separate the contribution from the direct light. An improved expression that accurately calculates the total fluence at the cavity wall as a function of light source location, cavity geometry and optical properties is determined based on theoretical and phantom studies. The theoretical study includes an expression for light fluence rate in an elliptical geometry instead of the spheroid geometry used previously. The calculated light fluence is compared to the measured fluence in patients of different cavity geometries and optical properties. The result can be used as a clinical guideline for future pleural PDT treatment.
doi:10.1117/12.2004941
PMCID: PMC4437736  PMID: 25999646
Light dosimetry; photodynamic therapy; HPPH; light fluence
21.  Parameter determination for BPD mediated vascularPDT 
The cell killing mechanism of benzoporphyrin derivative monoacid ring A (BPD) is known to be predominantly apoptotic or vascular, depending on the drug-light interval (DLI). With a 3 hour DLI, necrosis develops secondary to tumor cell damage, while with a 15 minute DLI, necrosis results from treatment-created vascular damage. The purpose of this study is to examine if the different mechanisms of cell death will affect the photochemical parameters for the macroscopic singlet oxygen model. Using the RIF model of murine fibrosarcoma, we determined the four photochemical parameters (ξ, σ, β, γ) and the threshold singlet oxygen dose for BPD-mediated PDT through evaluation of the extent of tumor necrosis as a function of PDT fluence rate and total fluence. Mice were treated with a linear source at fluence rates from 12–150 mW/cm and total fluences from 24–135 J/cm. BPD was administered at 1mg/kg with a 15 minute DLI, followed by light delivery at 690nm. Tumors were excised at 24 hours after PDT and necrosis was analyzed via H&E staining. The in-vivo BPD drug concentration is determined to be in the range of 0.05–0.30 μM. The determination of these parameters specific for BPD and the 15 minute DLI provides necessary data for predicting treatment outcome in clinical BPD-mediated PDT. Photochemical parameters will be compared between 1mg/kg DLI 3 hours and 1mg/kg DLI 15 minutes.
doi:10.1117/12.2040305
PMCID: PMC4437733  PMID: 25999653
22.  Effect of 630-NM pulsed laser irradiation on the proliferation of HeLa cells in Photofrin®-mediated photodynamic therapy 
Laser Therapy  2011;20(2):135-138.
Background and Aims: Red laser light of wavelength 630 nm is usually used for Photofrin®-mediated photodynamic therapy (PDT). The 630-nm light employed in PDT corresponds to the region of the wavelength used in low-level laser therapy (LLLT) may influence on the photodynamic effect required for killing cancer cells. The aim of this in vitro study was to investigate the changes in cell viability and degree of cell proliferation after Photofrin®-mediated PDT using 630-nm pulsed laser irradiation (10 Hz repetition rate and 7–9 ns pulse width), which was clinically found to induce no remarkable cell injury.
Materials and Methods: A study has been conducted in which HeLa cells are incubated with Photofrin® for 15 min (10 µg/ml). Irradiation was carried out at an average fluence rate of 50 mW/cm2 with light doses of 1, 3, and 5 J/cm2. The cytotoxic effects on the cells are evaluated by the XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) assay.
Results: The results showed that the laser irradiated cells exhibited a greater clonogenic activity than normal and PDT treated cells for a short period after the laser irradiation.
Conclusion: If the level of 630-nm pulsed laser irradiation employed in a PDT is comparatively lowered, it would have a biostimulatory effect like that of in LLLT.
doi:10.5978/islsm.20.135
PMCID: PMC3799027  PMID: 24155522
PDT; LLLT; HeLa cells; Cell proliferation; 630-nm pulsed laser
23.  Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy 
Journal of Biomedical Optics  2013;18(10):101314.
Abstract.
We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.
doi:10.1117/1.JBO.18.10.101314
PMCID: PMC3726228  PMID: 23897439
photodynamic therapy; neutrophil elastase; neutrophil elastase 680 FAST; molecular imaging; in vivo imaging; immune cell imaging; photoactivatable probe; confocal microscopy; protease activity; Photofrin; 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a
24.  Susceptibility of Candida Species to Photodynamic Effects of Photofrin 
The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.
doi:10.1128/AAC.48.6.2000-2006.2004
PMCID: PMC415596  PMID: 15155191
25.  Efficacy of 5-aminolevulinic acid-mediated photodynamic therapy using light-emitting diodes in human colon cancer cells 
Oncology Reports  2013;29(3):911-916.
5-Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) (ALA-PDT) is a highly selective treatment for malignant cells. ALA-PDT has the potential to develop into a novel therapeutic strategy for various types of cancer. Recently, light-emitting diodes (LEDs), which are inexpensive, stable and easier to handle compared to lasers, have been used in PDT as a light source. However, in colorectal cancer (CRC), the efficacy of ALA-PDT in combination with LEDs has not been fully assessed. Therefore, in this study, we evaluated the antitumor effect of ALA-PDT using various LEDs in colon cancer cells. The HT-29 human colon cancer cell line was used both in vitro and in vivo. HT-29 cells were seeded in 96-well plates. Following 5-ALA administration, cells were irradiated using LEDs at different wavelengths. Three types of LEDs, blue (peak wavelength, 456 nm), white (broad-band) and red (635 nm) were used. Twenty-four hours after irradiation, the cytotoxic effects of ALA-PDT were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In order to evaluate the antitumor effect of ALA-PDT in vivo, nude mice were inoculated with HT-29 cells. Xenograft mice were injected intraperitoneally with 5-ALA and irradiated with 3 types of LEDs at a measured fluence rate of 96 mW/cm2 and fluence of 32 J/cm2. Each group comprised 6 mice. ALA-PDT was repeated 3 times at weekly intervals. Tumor weights were measured. Compared to the controls, ALA-PDT using LEDs showed significant antitumor effects in vitro and in vivo. The blue and white LEDs demonstrated greater antitumor effects compared to the red LEDs in vitro and in vivo. In particular, tumor inhibition rates in the blue and white LED groups were approximately 88% to those of the control group in the mouse models. In conclusion, ALA-PDT using LEDs is effective and useful in the treatment of CRC cells. This method could be a novel treatment modality for CRC.
doi:10.3892/or.2013.2220
PMCID: PMC3597538  PMID: 23291627
5-aminolevulinic acid; photodynamic therapy; colon cancer; light-emitting diode; protoporphyrin IX

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