Wavelength-dependent properties of photodynamic therapy using hypericin in vitro and in an animal model

Wavelength effects in photodynamic therapy (PDT) with hypericin (HY) were examined in a C26 colon carcinoma model both in vitro and in vivo. Irradiation of HY-sensitized cells in vitro with either 550 or 590 nm caused the loss of cell viability in a drug- and light-dose-dependent manner. The calculated ratio of HY-based PDT (HY-PDT) efficiencies at these two wavelengths was found to correlate with the numerical ratio of absorbed photons at each wavelength. In vivo irradiation of C26-derived tumors, 6 h after intraperitoneal administration of HY (5 mg/kg), caused extensive vascular damage and tumor necrosis. The depth of tumor necrosis (d) was more pronounced at 590 than at 550 nm and increased when the light dose was raised from 60 to 120 J/cm2. The maximal depths of tumor necrosis (at 120 J/cm2) were 7.5+/-1.5 mm at 550 nm and 9.9+/-0.8 mm at 590 nm. Both values are rather high in view of the limited penetration of green-yellow light into the tissue. Moreover, the depth ratio, d590/d550 = 1.3 (P < 0.001), is smaller than expected considering the 2.2-fold lower HY absorbance and the 1.7-fold lower tissue penetration of radiation at 550 than at 590 nm. This finding indicates that in vivo the depth at which HY-PDT elicits tumor necrosis is not only determined by photophysical considerations (light penetration, number of absorbed photons) but is also influenced significantly by other mechanisms such as vascular effects. Therefore, despite the relatively short-wavelength peaks of absorption, our observations suggest that HY is an effective photodynamic agent that can be useful in the treatment of tumors with depths in the range of 1 cm.     

PEG-m-THPC-mediated photodynamic effects on normal rat tissues

Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non-tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.     

PHOTOBLEACHING OF PORPHYRINS USED IN PHOTODYNAMIC THERAPY AND IMPLICATIONS FOR THERAPY

Abstract— The development of an extraction procedure to quantitate dihematoporphyrin ether (DHE) concentration in tissues correlated to fluorescence measurements from instrumentation developed for in vivo fluorimetry was examined. In vivo fluorometric results from mouse mammary carcinoma (SMT-F) were calibrated against results of the chemical extraction assay quantitated spectrophotometrically. Fluorescence and drug extractable levels increase in a linear fashion with injected dose. Loss of porphyrin fluorescence (photobleaching) and intra-tumoral porphyrin level has been demonstrated both in vitro (NHIK cells) and in vivo (SMT-F tumor) during illumination with light following exposure to Hpd or DHE. This process is essentially independent of porphyrin tumor level in vivo and could lead to tumor protection at very low porphyrin levels. On the other hand, this photobleaching process which occurs concurrent with cellular inactivation and tissue damage due to the photodynamic process can be exploited to protect normal tissue during photodynamic therapy (PDT) and thus greatly enhance the therapeutic ratio. This has been demonstrated in patients undergoing PDT.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

Damage Threshold of Normal Rat Brain in Photodynamic Therapy

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10¹⁶ photons/cm³. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.     

DRUG and LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR and NORMAL TISSUE RESPONSE

Abstract It is clinically relevant to determine drug and light dose combinations where complete tumor response is accompanied by little or no photosensitivity, and minimal damage to normal tissues. Although reciprocity of RIF tumor cell clonogenicity has been established within a range of drug and light doses, no quantitative data exist for reciprocity of tumor response. This study has examined reciprocity of drug and light doses for tumor response and normal tissue damage in two experimental mouse models. Representative tumors were examined for vascular damage after treatment. Reciprocity of drug and light doses for tumor response was observed over a range of drug/light combinations in both tumor models. Reciprocity failed when drug dose was reduced below a threshold value. For reciprocal drug/light combinations, complete vascular stasis occurred in the tumor and surrounding skin which was followed by necrosis of those tissues. In non-reciprocal PDT combinations, there was vascular damage to the tumor but no damage to the surrounding normal tissues. Tumors responded initially, but no cure was obtained. Tumor cure was only observed under conditions where a considerable margin of normal tissue surrounding the tumor was damaged. This conclusion was supported by shielding experiments done to assess the contribution of normal tissue damage to tumor response. Reciprocity of drug and light doses for tumor response was therefore shown to exist only at high drug doses, which were not low enough to reduce skin photosensitivity in our models.     

Photodynamic Therapy of Human Glioma (U87) in the Nude Rat

Abstract— We measured the response of normal brain and the human U87 glioma implanted in the brain of rats (n = 65) to photodynamic therapy (PDT) using Photofrin as the sensitizer. Normal brain and U87 tumor implanted within brain of athymic (nude) rats were subjected to PDT (12.5 mg/kg of Photofrin) at increasing optical energy doses (35 J/cm², 140 J/cm², 280 J/cm²) of 632 nm light. Photofrin concentration in tumor, brain adjacent to tumor and normal brain were measured in a separate population of rats. Twenty-four hours after PDT, the brains were removed, sectioned, stained with hematoxylin and eosin (H&E), and the volumes of the PDT-induced lesion measured. Photofrin concentration in tumor greatly exceeded that of normal brain and brain adjacent to tumor (>20×). Both normal brain and U87 tumor exhibited superficial tissue damage with PDT at 35 J/cm². However, both normal and tumor-implanted brain exhibited tissue damage with increasing optical dose. A heterogeneous pattern of pannecrosis along with a uniform volume of pannecrosis was detected in the tumor. In contrast, normal brain exhibited a uniform sharply demarcated volume of necrosis. Our data indicate that the U87 human brain tumor model and the normal brain in the athymic rat are sensitive to PDT and Photofrin with an optical dose-dependent response to treatment.     

DRUG AND LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR CELL CLONOGENICITY AND HISTOLOGIC CHANGES

Abstract— The dependence of photodynamic therapy (PDT) on changes in drug and light doses was determined in C3H/HeJ mice bearing the RIF tumor. Measurements of tumor clonogenicity were determined 24 h after PDT over a range of drug and light doses. Representative histological samples were prepared at each of these doses. Both the drug and light dose dependence experiments showed an exponential decrease in clonogenicity after an initial shoulder region. Reciprocity of drug and light dose was established from those clonogenicity curves. Histological examination of tumors gave information concerning the localization of gross damage within tumors. Increases of light dose in PDT were shown to extend the depth of necrosis within tumors. Increases of drug dose produced enlargements in the area of necrotic spots produced by PDT     

Organic Nanoparticles with Persistent Luminescence for In Vivo Afterglow Imaging-Guided Photodynamic Therapy

Optical imaging-guided photodynamic therapy (PDT), with precise localization and non-invasive treatment of tumors, is an emerging technique with great potential for cancer therapy. However, impaired by tissue auto-fluorescence that causes low signal-to-background ratio (SBR), most fluorescence imaging systems show poor sensitivity to tumors in vivo. In this study, we synthesized organic nanoparticles (ONPs) with persistent luminescence and good biocompatibility for afterglow imaging-guided PDT. The ONPs displayed near-infrared light emission with half-life time at minute level, which offered high SBR and good tissue penetration for in vivo afterglow tumor imaging. Taking advantage of their abundant singlet oxygen generation by NIR laser irradiation guided to the tumor sites, the ONPs also enabled imaging-guided PDT for efficient suppression of tumor growth in mice with minimal damage to major organs.     

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.     

In vitro toxicity testing of zinc tetrasulfophthalocyanines in fibroblast and keratinocyte cells for the treatment of melanoma cancer by photodynamic therapy

A series of water-soluble tetrasulfonated metallophthalocyanines (MPcs) dyes have been studied to be used as a drug or photosensitizer (PS) in photodynamic therapy (PDT) for the treatment of cancers. During PDT the PS is administrated intravenously or topically to the patient before laser light at an appropriate wavelength is applied to the cancerous area to activate the PS. The activated PS will react with oxygen typically present in the cancerous tissue to generate reactive oxygen species for the destruction of the cancerous tissue. This in vitro study aimed at investigating the cytotoxic effects of different concentrations of zinc tetrasulfophthalocyanines (ZnTSPc) activated with a diode laser (λ = 672 nm) on melanoma, keratinocyte and fibroblast cells. To perform this study 3 × 10? cells/ml were seeded in 24-well plates and allowed to attach overnight, after which cells were treated with different concentrations of ZnTSPc. After 2h, cells were irradiated with a constant light dose of 4.5J/cm2. Post-irradiated cells were incubated for 24 h before cell viability was measured using the CellTiter-Blue Viability Assay. Data indicated high concentrations of ZnTSPc (60-100 μg/ml) in its inactive state are cytotoxic to the melanoma cancer cells. Also, results showed that photoactivated ZnTSPc (50 μg/ml) was able to reduce the cell viability of melanoma, fibroblast and keratinocyte cells to 61%, 81% and 83% respectively. At this photosensitizing concentration the efficacy the treatment light dose of 4.5J/cm2 against other light doses of 2.5J/cm2, 7.5J/cm2 and 10J/cm2 on the different cell lines were analyzed. ZnTSPc at a concentration of 50 μg/ml activated with a light dose of 4.5J/cm2 was the most efficient for the killing of melanoma cancer cells with reduced killing effects on healthy normal skin cells in comparison to the other treatment light doses. Melanoma cancer cells after PDT with a photosensitizing concentration of 50μg/ml and a treatment light dose of 4.5J/cm2 showed certain apoptosis characteristics such as chromatin condensation and fragmentation of the nucleus. This concludes that low concentrations of ZnTSPc activated with the appropriate light dose can be used to induce cell death in melanoma cells with the occurrence of minimal damage to surrounding healthy tissue.     

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons

Photodynamic therapy (PDT) is a clinical treatment in which a light‐absorbing drug called a photosensitizer (PS) is combined with light and molecular oxygen to generate cytotoxic singlet oxygen. PDT provides additional tissue selectivity compared to conventional chemotherapy as singlet oxygen is generated only in areas in which PS accumulates and that are simultaneously illuminated by a light source with sufficient irradiance and dose. Early PDT beacons built on this concept by adding an analyte‐responsive element that simultaneously turns on PDT and fluorescence, providing both an additional layer of selectivity and real‐time feedback of the PS′s activation state. More recent PDT beacons have expanded this idea, with new methods now available for sensing analytes, generating singlet oxygen, and reporting treatment status. In this Minireview, we consider developments in advanced activation strategies implemented in therapeutic and theranostic beacons.     

DH-I-180-3-mediated photodynamic therapy: biodistribution and tumor vascular damage

An important goal of photodynamic therapy (PDT) for treatment of various cancers is to shorten PDT-performing time and simultaneously enhance PDT efficacy. Here, we investigated the nontumor tissue distribution of and the tumor vascular damage caused by a new photosensitizer, DH-I-180-3, in mice with implanted EMT6 mammary tumor cells. In addition, we performed cell-based assays to evaluate the basic antitumor effect of DH-I-180-3/PDT in EMT6 cells. After administration of PDT, the type of cell death was characterized to be apoptosis, and a change in the mitochondrial membrane potential was also observed within minutes. On the other hand, tumor growth was remarkably retarded in vivo in mice that received DH-I-180-3/PDT, compared with mice in the control group, which were exposed to light irradiation alone. Finally, tumors in some mice nearly healed. The antitumor drug reached a maximum concentration approximately 3 h after administration. However, PDT was most effective when there was substantial accumulation of DH-I-180-3 in the tumor vasculature and in healthy tissue. The histological demonstration provided further evidence of tumor vascular damage. On the basis of these findings, we suggest that PDT with the photosensitizer DH-I-180-3 induces vascular damage with blood vessel shutdown, in addition to direct killing of tumor cells, in mice.     

Evaluation of the photosensitizer Tookad for photodynamic therapy on the Syrian golden hamster cheek pouch model: light dose, drug dose and drug-light interval effects

We have evaluated the efficacy of the new photosensitizer (PS) Tookad in photodynamic therapy (PDT) in vivo. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 762 and 538 nm. The light dose, drug dose and drug injection-light irradiation interval (DLI), ranging between 100 and 300 J/cm2, 1 and 5 mg/kg and from 10 to 240 min, respectively, were varied, and the response to PDT was analyzed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. The level of PDT response, macroscopically and histologically, shows a strong dependence on the DLI, light dose and drug dose at the applied conditions in the normal hamster cheek pouch. A decay of the tissular response with increasing DLI is observed corresponding to a time of half-maximum response ranging from 10 to 120 min, depending on drug dose and light dose. The tissues affected at the lowest doses are predominantly the vascularized diffuse connective tissue situated between the inner and outer striated muscle (SM) layers as well as these muscle layers themselves. The highest response at the shortest DLI and the absence of a measurable response at DLI longer than 240 min at 300 J/cm2 and drug dose of 5 mg/kg are characteristics of a predominantly vascular effect of this PS. This observation suggests that Tookad could be effective in PDT of vascularized lesions or pathologies associated with the proliferation of neovessels.     

Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.     

Reactive Oxygen Species Explicit Dosimetry for Photofrin-mediated Pleural Photodynamic Therapy

Explicit dosimetry of treatment light fluence and implicit dosimetry of photosensitizer photobleaching are commonly used methods to guide dose delivery during clinical PDT. Tissue oxygen, however, is not routinely monitored intraoperatively even though it is one of the three major components of treatment. Quantitative information about in vivo tissue oxygenation during PDT is desirable, because it enables reactive oxygen species explicit dosimetry (ROSED) for prediction of treatment outcome based on PDT-induced changes in tumor oxygen level. Here, we demonstrate ROSED in a clinical setting, Photofrin-mediated pleural photodynamic therapy, by utilizing tumor blood flow information measured by diffuse correlation spectroscopy (DCS). A DCS contact probe was sutured to the pleural cavity wall after surgical resection of pleural mesothelioma tumor to monitor tissue blood flow (blood flow index) during intraoperative PDT treatment. Isotropic detectors were used to measure treatment light fluence and photosensitizer concentration. Blood-flow-derived tumor oxygen concentration, estimated by applying a preclinically determined conversion factor of 1.5 × 10⁹ μMs cm⁻² to the blood flow index, was used in the ROSED model to calculate the total reacted reactive oxygen species [ROS]rx. Seven patients and 12 different pleural sites were assessed and large inter- and intrapatient heterogeneities in [ROS]rx were observed although an identical light dose of 60 J cm⁻² was prescribed to all patients.     

Photodynamic therapy with chlorin e6. A morphologic study of tumor damage efficiency in experiment.

Morphological changes in rat sarcoma M-1 after photodynamic treatment with chlorin e6 were studied. The frequency of necrosis appearance and the depth of its spreading in tumor tissue were evaluated after intraperitoneal injection of chlorin e6 in doses of 1-10 mg kg-1 and subsequent irradiation by a krypton laser with light energy density 22.5-135 J cm-2, using the method of vital staining with Evans blue. It was found that the antitumoral effect of photodynamic treatment was strengthened by increasing the dose of the agent and light and reduced by increasing the time interval between chlorin e6 injection and light irradiation. The treatment being given in the parameters mentioned produced a depth of tumor necrosis which varied from 4.0 mm to 16.6 mm. The mechanisms of tumor tissue damage after photodynamic treatment in vivo are discussed.     

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monte Carlo Model of Stricture Formation in Photodynamic Therapy of Normal Pig Esophagus

Photodynamic therapy (PDT) is FDA-approved for use in patients with Barrett's esophagus using porfimer sodium (2 mg per kg) and a recommended light dose of 130 J cm⁻¹ for high grade dysplasia. Despite uniform drug and light doses, the clinical outcome of PDT is variable. A significant number of PDT cases result in esophageal strictures, a side effect related to excessive energy absorption. The purpose of this project was to model esophageal stricture formation with a Monte Carlo simulation. An original multilayer Monte Carlo computer simulation was developed for esophageal PDT. Optical absorption and scattering coefficients were derived for mucosal and muscle layers of normal porcine esophagus. Porfimer sodium was added to each layer by increasing the absorption coefficient by the appropriate amount. A threshold-absorbed light dose was assumed to be required for stricture formation and ablation. The simulation predicted irreversible damage to the mucosa with a 160 J cm⁻¹ light dose and damage to the muscle layer with an additional 160 J cm⁻¹ light dose for a tissue porfimer sodium content of 3.5 mg kg⁻¹. The simulation accurately modeled photodynamic stricture formation in normal pig in vivo esophageal tissue. This preliminary work suggests that the absorbed light threshold for stricture formation may be between 2 and 4 J per gram of tissue.