In vivo photodynamic characteristics of the near-infrared photosensitizer 5,10,15,20-tetrakis(M-hydroxyphenyl) bacteriochlorin

This paper describes the photodynamic characteristics of the new near-infrared photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (mTHPBC or SQN400) in normal rat and mouse tissues. A rat liver model of photodynamic tissue necrosis was used to determine the in vivo action spectrum and the dose-response relationships of tissue destruction with drug and light doses. The effect of varying the light irradiance and the time interval between drug administration and light irradiation on the biological response was also measured in the rat liver model. Photobleaching of mTHPBC was measured and compared with that of its chlorine analog (mTHPC) in normal mouse skin and an implanted mouse colorectal tumor. The optimum wavelength for biological activation of mTHPBC in rat liver was 739 nm. mTHPBC was found to have a marked drug-dose threshold of around 0.6 mg kg-1 when liver tissue was irradiated 48 h after drug administration. Below this administered drug dose, irradiation, even at very high light doses, did not cause liver necrosis. At administered doses above the photodynamic threshold the effect of mTHPBC-PDT was directly proportional to the product of the drug and light doses. No difference in the extent of liver necrosis produced by mTHPBC was found on varying the light irradiance from 10 to 100 mW cm-2. The extent of liver necrosis was greatest when tissue was irradiated shortly after mTHPBC administration and necrosis was absent when irradiation was performed 72 h or later after drug administration, suggesting that the drug was rapidly cleared from the liver. In vivo photobleaching experiments in mice showed that the rate of bleaching of mTHPBC was approximately 20 times greater than that of mTHPC. It is argued that this greater rate of bleaching accounts for the higher photodynamic threshold and this could be exploited to enhance selective destruction of tissues which accumulate the photosensitizer.     

Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.     

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.     

Effects of tritiated water on the DNA of plasmid pBR 322

In an attempt to shed light on the influence of tritiated water for DNA we have investigated the damage with a simple plasmid DNA, pBR 322. The survival of covalently closed circular (CCC DNA) form was directly followed by agarose of gel electrophoresis. It was found that the survival percentage of DNA in tritiated water was observed almost the same as the irradiation of X-rays at the same absorbed doses. For the irradiation of gamma-rays, on the other hand, the decay rate was larger than those of both tritiated water and X-rays. The yields percentage of the broken pieces of DNA in tritiated water, X-rays and gamma-rays were found to be 43, 38 and 33% at 10(4) rad of the absorbed dose. It may be considered that the degree of danger in tritiated water is quite larger than of gamma-rays. It was also found that the dose rate effect was not observed in the case of tritiated water, X-rays and gamma-rays irradiation.     

Preclinical evaluation of a novel water-soluble chlorin E6 derivative (BLC 1010) as photosensitizer for the closure of the neovessels

In the present study, photodynamic activity of a novel photosensitizer (PS), Chlorin e(6)-2.5 N-methyl-d-glucamine (BLC 1010), was evaluated using the chorioallantoic membrane (CAM) as an in vivo model. After intravenous (i.v.) injection of BLC 1010 into the CAM vasculature, the applicability of this drug for photodynamic therapy (PDT) was assessed in terms of fluorescence pharmacokinetics, i.e. leakage from the CAM vessels, and photothrombic activity. The influence of different PDT parameters including drug and light doses on the photodynamic activity of BLC 1010 has been investigated. It was found that, irrespective of drug dose, an identical continuous decrease in fluorescence contrast between the drug inside and outside the blood vessels was observed. The optimal treatment conditions leading to desired vascular damage were obtained by varying drug and light doses. Indeed, observable damage was achieved when irradiation was performed at light doses up to 5 J/cm(2) 1 min after i.v. injection of drug doses up to 0.5 mg/kg body weight(b.w.). However, when irradiation with light doses of more than 10 J/cm(2) was performed 1 min after injection of drug doses up to 2 mg/kg body weight, this led to occlusion of large blood vessels. It has been demonstrated that it is possible to obtain the desired vascular occlusion and stasis with BLC 1010 for different combinations of drug and/or light doses.     

THE THEORY OF PHOTODYNAMIC THERAPY DOSIMETRY: CONSEQUENCES OF PHOTO-DESTRUCTION OF SENSITIZER

Photodynamic dose is defined as the area under the curve of sensitizer level plotted as a function of light dose. This is a photochemical definition of dose. We will show that this definition is useful in predicting photobiological response. The photodestruction of sensitizer during photodynamic therapy is shown to result in an upper limit on the photodynamic dose which can be delivered by an unlimited light dose. This limit results in the opportunity to make total photodynamic dose uniform to considerable depths (one to two centimeters). The existence of thresholds for permanent tissue damage allows protection of normal tissue from the large light doses required to achieve this limiting dose deep in the tissue. Higher sensitizer levels in the tumor permit tumor destruction while the normal tissues are protected. A clinical trial to determine the proper level of injected dose necessary for these results is required. This theory of photodynamic therapy (PDT) dosimetry is tested in the DBA-SMT experimental mouse tumor system. Combinations of drug and light which are not reciprocal but are nearly equal by this theory are shown to give equivalent tumor control at seven days post treatment. Reciprocal combinations of drug and light fail to give equivalent results when they ae selected using the theory to choose a combination where reciprocity should fail.     

Perylene-Based Reactive Oxygen Species Supergenerator for Immunogenic Photochemotherapy against Hypoxic Tumors

Reactive oxygen species (ROS) can act as cytotoxic radicals to directly kill tumor cells and concurrently trigger immunogenic cell death (ICD) to efficiently achieve tumor therapy. Thus motivated, we herein present one perylene monoamide-based ROS supergenerator (PMIC-NC) that not only induces hypoxia-enhanced Type-I ROS burst aided by proton transients but also triggers Type-I/II ROS production by electron or energy transfer under near-infrared (NIR) light irradiation and also elicits a strong ICD effect. More interesting, the mitochondria- and lung-specific distribution of PMIC-NC also boosts the tumor therapeutic efficiency. As a result, PMIC-NC was employed for NIR-triggered photodynamic therapy, hypoxia-enhanced chemotherapy and also displayed robust immunogenicity for systemic tumor eradication. This work thus contributes one proof-of-concept demonstration of perylene as an integrated therapeutic platform for efficient immunogenic photochemotherapy against hypoxic tumors.     

EFFICACY OF PHOTODYNAMIC THERAPY ON ORIGINAL AND RECURRENT RAT MAMMARY TUMORS

Abstract— Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of ˜ 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [¹⁴C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.     

Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.     

Effects of photodynamic therapy on the absorption properties of disulphonated aluminum phthalocyanine in tumor-bearing mice

Time-resolved reflectance spectroscopy was performed on tumor-bearing mice, administered with disulphonated aluminum phthalocyanine (AlS(2)Pc, 5 mg/kg body weight), before, during and after photodynamic therapy. This allowed us to evaluate the absorption spectrum of AlS(2)Pc in vivo from 610 to 700 nm, and to investigate how the therapeutic irradiation affects it. Two tumor locations (intraderma on the back and intramuscular in the leg), and two uptake times (3 and 12 h) were considered. As already observed previously, the absorption spectrum of AlS(2)Pc in vivo is centered at 680-685 nm. The irradiation causes a blue-shift of the measured line shape, more or less marked depending on the experimental conditions. A reduction in absorption is also often observed upon illumination with therapeutic light doses.     

Graphene oxide noncovalent photosensitizer and its anticancer activity in vitro

Graphene oxide (GO) was investigated as a potential drug-delivery system due to its special properties and biocompatibility. Thus far, little has been done to use GO as a photosensitive drug-delivery system and to explore its anticancer activity in vitro in photodynamic therapy applications. Here, a novel GO-hypocrellin A (GO-HA) hybrid was prepared by a simple noncovalent method and its photodynamic activity was studied for the first time. The results showed that an efficient loading amount of HA on GO was as high as 1.0 mg mg(-1) and the stability of the hybrid was superior to that of the free hypocrellin A in aqueous solution. Furthermore, GO-HA can be excited by irradiation with light of appropriate wavelength to generate singlet oxygen, and in vitro experiments illustrated that GO-HA was efficiently taken up by tumor cells, and that light irradiation of such impregnated cells resulted in significant cell death. Thus, these properties of GO-HA could possibly make it especially promising for use in clinical photodynamic therapy.     

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     

IMPROVED SURVIVAL FROM INTRACAVITARY PHOTODYNAMIC THERAPY OF RAT GLIOMA

The effectiveness of intratumoral photoradiation in photodynamic therapy (PDT) using a polyporphyrin photosensitizer was studied in the RT-2 rat glioma model. One week after intracerebral implantation of RT-2 cells, experimental rats received a single i.p. injection of 2 mg/kg of Photofrin. After administration of the photosensitizer (48 h), the tumors were partially resected and the exposed cavity was irradiated with 15 J of laser light at a wavelength of 630 nm. Further treatment with a large craniectomy significantly enhanced rat survival. Control rats which received no photosensitizer but were treated with surgery, alone or in combination with laser irradiation, succumbed from early tumor recurrence. Photodynamic therapy without decompressive surgery resulted in hemorrhagic infarction of residual tumor and adjacent brain with focal cerebral edema which resulted in cerebral herniation and early death. Our results indicate that photodynamic therapy is effective in treating residual brain tumor but at the expense of brain tissue surrounding the tumor. Unless relieved, intracranial pressure from photodynamic therapy-associated cerebral edema in this animal model resulted in shortened survival.     

Cellular Uptake and Photodynamic Activity of Protein Nanocages Containing Methylene Blue Photosensitizing Drug

This study reports that photosensitizers encapsulated in supramolecular protein cages can be internalized by tumor cells and can deliver singlet oxygen intracellularly for photodynamic therapy (PDT). As an alternative to other polymeric and/or inorganic nanocarriers and nanoconjugates, which may also deliver photosensitizers to the inside of the target cells, protein nanocages provide a unique vehicle of biological origin for the intracellular delivery of photosensitizing molecules for PDT by protecting the photosensitizers from reactive biomolecules in the cell membranes, and yet providing a coherent, critical mass of destructive power (by way of singlet oxygen) upon specific light irradiation for photodynamic therapy of tumor cells. As a model, we demonstrated the successful encapsulation of methylene blue (MB) in apoferritin via a dissociation–reassembly process controlled by pH. The resulting MB-containing apoferritin nanocages show a positive effect on singlet oxygen production, and cytotoxic effects on MCF-7 human breast adenocarcinoma cells when irradiated at the appropriate wavelength (i.e. 633 nm).     

Treatment of Malignant Melanoma by High-Peak-Power 1064 nm Irradiation Followed by Photodynamic Therapy

Irradiation of B16 pigmented melanoma subcutaneously transplanted in C57 mice with a single 650 mj pulse (10 ns) of 1064 nm light from a Q-switched Nd: YAG laser caused instantaneous bleaching of the pigmented tissue. Visual and histological examination of the resulting gray-colored tumor revealed the breakdown of melanosomes with no detectable alteration of the normal and tumor-overlying skin. Histological examination of the irradiated tumor showed some degree of vascular damage; the depth of the photodamage was not affected by the successive delivery of three consecutive light pulses. The bleached tumor grew at a modestly slower rate but the high-peak-power (HPP) laser treatment did not affect the tumor concentration of a photodynamic sensitizer Si(IV)-naphthalocyanine (isoBO-SiNc) intravenously injected 24 h before Nd : YAG irradiation. Treatment of the B16 pigmented melanoma by photodynamic therapy (PDT: 1 mg/kg isoBO-SiNc, 300 mW/cm², 520 J/cm²) from a 774 nm diode laser immediately after the 1064 nm irradiation resulted in a 16 day delay of tumor regrowth, which was markedly longer than the delay (ca 6 days) obtained after PDT under identical conditions without the preirradia-tion. Thus, pretreatment of pigmented tumors with HPP 1064 nm light appears to enhance their susceptibility to conventional PDT. The tumor response was further enhanced by repeating the combined HPP/PDT treatment at an interval of 10 days (regrowth delay: 27 days), as well as by applying hyperthermia immediately after HPP/PDT (regrowth delay: ca 34 days).     

MECHANISMS OF CELL KILLING IN PHOTODYNAMIC THERAPY USING A NOVEL in vivo DRUG/in vitro LIGHT CULTURE SYSTEM

Photodynamic therapy of certain neoplasms has emerged as a promising form of cancer treatment. This type of therapy involves the exogenous administration of a photosensitizer with subsequent exposure to light. The ensuing photochemical reaction results in destruction of the tumor. Whether tumor cells are destroyed directly by the photodynamic treatment or indirectly as a result of destruction of the tumor microvascular bed is unknown. To address this question, methods were adapted to test whether combinations of a photosensitizer and light resulted in direct cell killing of precision cut tissue slices placed in culture. The major advantages of this culture system are that photosensitizers are administered in vivo, tissue slices produced in minutes, placed in culture medium, and irradiated in vitro. Any resulting cellular destruction occurs in the absence of a functioning vascular system and indicates that photodynamic therapy acts through a direct cell killing mechanism. Tissue slice viability was monitored by two standard methods: assay for intracellular potassium and morphological examination at the electron microscopic level. The effects of hematoporphyrin derivative and light were examined on tissue slices produced from a prostate adenocarcinoma transplanted into male Copenhagen rats. The data indicate that direct killing of tumor slices occurs and is dependent on the irradiation protocol used.     

INTRATUMOR TEMPERATURE MEASUREMENTS DURING PHOTODYNAMIC THERAPY

Abstract Photodynamic therapy has been under investigation as a form of cancer treatment for a number of years. This procedure uses a light source of 630 nm to photoactivate the drug, Photofrin II. Researchers in the past have reported temperature increases during photodynamic therapy, by measuring surface temperature of the tumor or a single point temperature within the tumors. Three temperature points within the tumors have been measured in this study, to quantify the temperature distribution within the lesion. These temperatures were measured for photodynamic therapy treated mice and control mice receiving an exposure to the treatment light without the drug. The use of a filtered xenon arc lamp for the 630 nm light source produced larger temperature increases and thermal gradients within the tumors, than when an Argon dye laser was employed. This temperature increase is due in part to the broad wavelength output of this filtered lamp. When this thermal effect is present during PDT treatment, researchers have observed the development of shock proteins resulting in the induction of thermotolerance and resistance to subsequence hyperthermia treatments. Using the filtered arc lamp, mice receiving photodynamic therapy treatments displayed consistently higher temperature increases than control mice. The use of an argon dye laser, with sufficient air cooling of the tumor, can eliminate this thermal effect. It has been demonstrated that the use of filtered lamps produce thermal effects which cannot be eliminated, demonstrating that lasers should be the primary source of light used to photoirradiate animals for photodynamic therapy studies. The intratumor temperature increases should be documented at multiple positions, to determine the amount of thermotolerance which can be induced. When photodynamic therapy is followed with a subsequent hyperthermia treatment, this induced thermotolerance can then be taken into consideration.     

Photodynamic therapy of nonmelanoma skin cancer with topical hypericum perforatum extract--a pilot study.

Hypericin, the photoactive compound of Hypericum perforatum, is probably the most powerful photosensitizer found in nature. This compound has shown high potency in the photodynamic treatment of tumor cells. However, there is only limited knowledge regarding the photodynamic effect of hypericin on nonmelanoma skin cancer cells. The aim of this prospective study was to investigate the efficacy of photodynamic therapy with topical application of an extract of H. perforatum in actinic keratosis, basal cell carcinoma (BCC) and morbus Bowen (carcinoma in situ). The study was carried out on 34 patients--eight with actinic keratoses (AKs), 21 with BCC and five with Bowen's disease. The extract of H. perforatum was applied on the skin lesions under occlusion and that was followed by irradiation with 75 J cm(-2) of red light 2 h later. The treatment was performed weekly for 6 weeks on average. The percentage of complete clinical response was 50% for AKs, 28% in patients with superficial BCC and 40% in patients with Bowen's disease. There was only a partial remission seen in patients with nodular BCCs. A complete disappearance of tumor cells was found in the histologic preparation of 11% of patients with superficial BCCs and 80% in the patients with Bowen's disease. All patients complained of burning and pain sensations during irradiation. Although the results of this first clinical trial could be regarded as disappointing, there are still possibilities for improvement. Better preparation of the lesions, enhancement of hypericin delivery and other types of light exposure procedures could significantly improve the clinical outcomes of this relatively inexpensive treatment modality.     

Morphological changes of tumor microvasculature following hematoporphyrin derivative sensitized photodynamic therapy

Abstract There is increasing evidence that the tumor microvasculature is affected during porphyrin photodynamic therapy. In the following study, the effect of hematoporphyrin derivative photodynamic therapy on tumor microvasculature was studied by electron microscopy. Urothelial tumors, implanted subcutaneously in rats, were exposed to red light (> 590 nm; 360 J cm^?2) 72 h after injection of hematoporphyrin derivative at a dose of 5 mg kg^?1 of body weight. Prior to sampling, in vivo perfusion was carried out using a polyvalent cation, lanthanum, in 3% glutaraldehyde to define the endocapillary layer of endothelial cells. Samples of tumors were collected at 0, 1,2 and 4 h after completion of photodynamic therapy. Histological changes in endothelial cells were evident immediately following completion of light exposure. Immediate morphological changes included absence of the endocapillary layer as well as mitochondrial degeneration. The changes within tumor cells followed the changes within the microvasculature. This study indicates that the endothelial cell of tumor tissue is an important target of photodynamic therapy and may be responsible for the blood flow changes reported previously.     

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.