Barbara Krammer

Photophysics and photochemistry of photodynamic therapy: fundamental aspects

Photodynamic therapy (PDT) is a treatment modality for cancer and various other diseases. The clinical protocol covers the illumination of target cells (or tissue), which have been loaded with a photoactive drug (photosensitizer). In this review we describe the photophysical and primary photochemical processes that occur during PDT. Interaction of light with tissue results in attenuation of the incident light energy due to reflectance, absorption, scattering, and refraction. Refraction and reflection are reduced by perpendicular light application, whereas absorption can be minimized by the choice of a photosensitizer that absorbs in the far red region of the electromagnetic spectrum. Interaction of light and the photosensitizer can result in degradation, modification or relocalization of the drug, which differently affect the effectiveness of PDT. Photodynamic therapy itself, however, employs the light-induced chemical reactions of the activated photosensitizer (triplet state), resulting in the production of various reactive oxygen species, amongst them singlet oxygen as the primary photochemical product. Based on these considerations, the properties of an ideal photosensitizer for PDT are discussed. According to the clinical experience with PDT, it is proposed that the innovative concept of PDT is most successfully implemented into the mainstream of anticancer therapies by following an application-, i.e. tumor-centered approach with a focus on the actual clinical requirements of the respective tumor type.

Apoptosis following photodynamic tumor therapy: induction, mechanisms and detection.

As a treatment modality for malign and certain non-malignant diseases, photodynamic therapy (PDT) involves a two step protocol which consists of the (selective) uptake and accumulation of a photosensitizing agent in target cells and the subsequent irradiation with light in the visible range. Reactive oxygen species (ROS) produced during this process cause cellular damage and, depending on the treatment dose/severity of damage, lead to either cellular repair/survival, apoptotic cell death or necrosis. PDT-induced apoptosis has been focused on during the last years due to the intimate connection between ROS generation, mitochondria and apoptosis; by this PDT employs mechanisms different to those in the action of radio- and chemotherapeutics, giving rise to the chance of apoptosis induction by PDT even in cells resistant to conventional treatments. In this review, the (experimental) variables determining the cellular response after PDT and the known mechanistic details of PDT-triggered induction and execution of apoptosis are discussed. This is accompanied by a critical evaluation of wide-spread methods employed in apoptosis detection with special respect to in vitro/cell-based methodology.

Comparative in vitro study on the characteristics of different photosensitizers employed in PDT.

At present a wide range of photosensitizers are employed in photodynamic therapy (PDT) that have very different characteristics. Although, countless in vitro studies on the attributes of photosensitizers do exist, a direct comparison of these substances on one cell line are rare and may contribute to the choice of the optimal photoactive substance for a specific application. We therefore evaluated the properties of six widespread photosensitizers, namely Foscan, Fospeg, hypericin, aluminum (III) phthalocyanine tetrasulfonate chloride (AlPcS(4)), 5-aminolevulinic acid (ALA), and Photofrin in terms of: (i) cytotoxicity without illumination, (ii) phototoxicity, (iii) cellular uptake and release, and (iv) apoptosis induction in A431 human epidermoid carcinoma cells using comparable illumination regimens. We clearly show that meso-tetrahydroxyphenylchlorin (mTHPC, Foscan) is a very effective photosensitizer inducing high phototoxicity at very low concentrations. Similar in vitro characteristics and phototoxicity were observed for Fospeg, the water-soluble formulation of mTHPC. Hypericin, a photosensitizer extracted from plants of the Hypericum genus, is very effective in inducing apoptosis over a wide range of light fluences. AlPcS(4) absorbs light of 674 nm wavelength providing a higher penetration depth in tissue. Its hydrophilic character allows for application as aqueous solution. ALA can be administered at very high concentrations without producing cytotoxic effects in the dark. The intracellular concentration of protoporphyrin IX rapidly decreases after withdrawal of ALA, thus minimizing the period of light sensitivity post PDT. Among all photosensitizers Photofrin has most clinical approvals and serves as standard.

Cellular Mechanisms and Prospective Applications of Hypericin in Photodynamic Therapy

During the last decades, Photodynamic Therapy (PDT) has been established as a powerful alternative approved by health agencies of several countries for treatment of various malignant and some non-malignant diseases. PDT makes use of the light-induced destruction of target cells by formation of cytotoxic products in the presence of a photosensitizing agent and oxygen. The light-dependent tumor destructive properties of Hypericin have drawn attention to its promising application as a photosensitizer in the frame of PDT. Hypericin is a naturally occurring secondary metabolite in plants of the Hypericum genus, with Hypericum perforatum (St. Johns wort) as it is a commonly known representative. This review focuses on the cellular mechanisms of Hypericin-based phototoxicity and provides an outlook for future application of Hypericin as a fluorescing and photosensitizing agent for diagnosis and treatment of cancerous diseases, respectively.

Comparative characterization of the efficiency and cellular pharmacokinetics of Foscan®- and Foslip®-based photodynamic treatment in human biliary tract cancer cell lines

Due to the poor prognosis and limited management options for perihilar cholangiocarcinoma (CC) the development of alternatives for treatment is an important topic. Photodynamic therapy (PDT) with porfimer as palliative or neoadjuvant endoscopic treatment of non-resectable perihilar CC has improved quality of life and survival time, but cannot eradicate the primary tumors because of inadequate tumoricidal depth (4 mm only around the tumor stenoses). The use of meta-tetrahydroxyphenyl chlorin (mTHPC) and photoactivation at higher wavelengths (650-660 nm) provides high tumoricidal depth (10 mm) for PDT of pancreatic cancer and should yield similar tumoricidal depth in CC. This study investigates the photodynamic characteristics of mTHPC in solvent-based formulation (Foscan) and in liposomal (water soluble) formulation (Foslip) in an in vitro model system consisting of two biliary cancer cell lines (GBC, gall bladder cancer and BDC, bile duct cancer cells). Dark toxicity, photodynamic efficiency, time-dependent uptake and retention and intracellular localization of Foscan and Foslip were studied. The results prove mTHPC as a potent photosensitizing agent with high phototoxic potential in biliary cancer cells as a concentration of 600 ng ml(-1) and irradiation with 1.5 J cm(-2) (660 +/- 10 nm) is sufficient for about 90% cell killing. Addition of foetal bovine serum (FBS) to the incubation medium and analysis of the uptake and phototoxic properties reveals that both photosensitizer formulations bind to serum protein fractions, i.e. no difference between Foscan and Foslip can be found in the presence of FBS. Laser scanning fluorescence microscopy indicates a similar pattern of perinuclear localization of both sensitizers. This study demonstrates the potential of mTHPC for treatment of bile duct malignancies and provides evidence that Foslip is an equivalent water-soluble formulation of mTHPC that should ease intravenous application and thus clinical use of mTHPC.

Characterization of the cell death modes and the associated changes in cellular energy supply in response to AlPcS4-PDT

Photodynamic therapy (PDT) can result in both types of cell death, apoptosis or necrosis. Several steps in the induction and execution of apoptosis depend on ATP and the intracellular ATP level has been shown to be one determinant in whether apoptosis or necrosis occurs. Therefore, photochemical damage of cellular targets involved in energy supply might play a crucial role in the mode of cell death being executed. The present study is aimed at the characterization of changes in cellular energy supply and the associated cell death modes in response to PDT. Using the human epidermoid carcinoma cell line A431 and aluminium(III) phthalocyanine tetrasulfonate chloride (2.5 microM) as a photosensitizer, we studied the changes in mitochondrial function and intracellular ATP level after irradiation with different light doses. Employing assays for caspase-3 activation and nuclear fragmentation, 50% of the cells were found to undergo apoptosis after irradiation between 2.5 to 3.5 J cm(-2) while the remainder died by necrosis. At higher light doses (> 6 J cm(-2)), neither caspase-3 activation nor nuclear fragmentation was observed and this suggests that these cells died exclusively by necrosis. Necrotic cell death was also associated with a rapid decline in mitochondrial activity and intracellular ATP. By contrast, with apoptosis the loss of mitochondrial function was delayed and the ATP level was maintained at near control levels for up to eight hours which was far beyond the onset of morphological changes. These data suggest that, depending on the light dose applied, both, necrosis as well as apoptosis can be induced with AlPcS4 mediated PDT and that photodamage in energy supplying cellular targets may influence the mode of cell death. Further, it is speculated that cells undergoing apoptosis in response to PDT might maintain a high ATP level long enough to complete the apoptotic program.

The Modes of Cell Death Induced by PDT: An Overview

Summary Photodynamic therapy (PDT) is a relatively new treatment for malignant and non-malignant diseases and by now successfully employed in many clinical applications. It is typically carried out as a two-step protocol with target cells first being selectively loaded with a photosensitizer followed by irradiation with light of the appropriate wavelength. Subsequent photochemical reactions lead to the production of reactive oxygen species (ROS) and cell death. PDT can trigger both modes of cell death, apoptosis and necrosis in target cells. Apoptosis is an active, controlled and energy-requiring process and therefore contrasts necrosis, which is an entropic event and in most cases a consequence of loss of membrane integrity and metabolic homeostasis. Photodynamic therapy has been shown to effectively induce apoptosis in several model systems since ROS formed by photoprocesses can directly damage mitochondria, which act as key regulators in active cell death. Therefore PDT may be able to set off the apoptotic cascade even in those cells which were shown to be resistant to apoptosis in response to chemotherapy or ionizing radiation. The cell death mode after PDT is of interest since it influences the response of the immune system and therefore the effectiveness of the treatment: apoptotic as well as necrotic cell death influence the activity and the specific response of various cell types involved in potential antitumor response of the immune system. The mode of cell death triggered by PDT can be influenced by altering the treatment protocol and can lead to a desired apoptosis/ necrosis ratio most advantageous for complete tumor eradication.

Does δ-aminolaevulinic acid induce genotoxic effects?

Abstract 5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway for haem. The presence of exogenous ALA bypasses the feedback control and may induce the accumulation of PpIX. Since haem-containing enzymes are essential for energy metabolism, every nucleated cell in the body must have at least a minimal capacity to synthesize PpIX. Photodynamic therapy (PDT), which is the treatment of malignant lesions with light following the administration of a tumour-localizing photosensitizers leads to oxidative damage, including the formation of genotoxic membrane degradation products via lipid peroxidation. In addition, it has been demonstrated that ALA itself can form the reactive oxygen species O2, H2O2 and OH− by auto-oxidation, suggesting that it could potentially induce DNA damage. Therefore cultures of rat hepatocytes, which have been demonstrated to be very sensitive indicators for genotoxic effects induced by the lipid peroxidation product 4-hydroxynonenal and analogous aldehydes, were examined for possible mutagenic effects after treatment with ALA in the absence of light. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Compared with the controls, significantly elevated levels of chromosomal aberrations and micronuclei were observed at concentrations of 1 μg ml1 or greater. Chromosomal aberrations and micronuclei were found to increase up to a concentration of 100 μg ml1 ALA. While micronuclei decrease at higher concentrations, chromosomal aberrations remain at the same level. The kinetics of PpIX formation after induction with 100 and 1000 μg ml−1 ALA appear to be the same for both concentrations, suggesting that the induction of chromosomal aberrations may be due to PpIX.

Role of calcium in photodynamically induced cell damage of human fibroblasts.

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca2+]i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer‐treated cells, the increase in [Ca2+]i is related to the amount of light and extracellular [Ca2+]e. The increase in [Ca2+]i was substantially reduced in the absence of [Ca2+]e. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca2+]i at high [Ca2+]e and was greater at low [Ca2+]e. Application of Ca2+ channel blockers, such as Co2+, Cd2+ or verapamil, could not prevent the increase of [Ca2+]i. Our results indicate that activation of the photosensitizer, AlPcS4, causes an influx of Ca2+, which protects cells from photodamage. At low [Ca2+]e and high fluence values, release of Ca2+ from internal stores probably as a protective measure occurs in order to increase the [Ca2+]i.

In-vitro investigation of ALA-induced protopoyphyrin IX

Protoporphyrin IX (PpIX) induced endogenously by delta-aminolevulinic acid (ALA), can be used to destroy photodynamically tumor cells. The influence of several parameters on the PpIX formation of human skin fibroblasts was investigated by fluorescence spectrophotometry. The PpIX formation increases (1) with the pH value of ALA (2) with the ALA incubation time in a moderate sigmoidal manner, and (3) with the ALA concentration up to 700 micrograms ml-1. Other parameters, such as cell washing procedures, have no influence on the PpIX production. ALA has to be applied in a concentration 30 times higher than external protoporphyrin IX and Photosan 3 in order to produce the same cytotoxic damage. Protoporphyrin bleaching and photoproduct generation at 646 nm was observed. Additional information about intracellular PpIX formation kinetics and its topographically correlation to cell structures was gained by a CCD camera mounted on a fluorescence microscope. A few minutes after the onset of incubation with ALA, PpIX generation is observed in the mitochondria, followed by relocalization in the cytoplasm and the nuclear membrane.