Photodynamic therapy involves administration of a tumor-localizing photosensitizing agent, which may require metabolic synthesis (i.e., a prodrug), followed by activation of the agent by light of a specific wavelength. This therapy results in a sequence of photochemical and photobiologic processes that cause irreversible photodamage to tumor tissues. Results from preclinical and clinical studies conducted worldwide over a 25-year period have established photodynamic therapy as a useful treatment approach for some cancers. Since 1993, regulatory approval for photodynamic therapy involving use of a partially purified, commercially available hematoporphyrin derivative compound (Photofrin) in patients with early and advanced stage cancer of the lung, digestive tract, and genitourinary tract has been obtained in Canada, The Netherlands, France, Germany, Japan, and the United States. We have attempted to conduct and present a comprehensive review of this rapidly expanding field. Mechanisms of subcellular and tumor localization of photosensitizing agents, as well as of molecular, cellular, and tumor responses associated with photodynamic therapy, are discussed. Technical issues regarding light dosimetry are also considered.

Photodynamic Therapy

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative, particularly in early stage tumors. It can prolong survival in patients with inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment. CA Cancer J Clin 2011;61:250-281. V C

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Photodynamic therapy of cancer: An update†‡

1.1 Photodynamic Therapy and Imaging
The purpose of this review is to present the current state of the role of imaging in photodynamic therapy (PDT). In order for the reader to fully appreciate the context of the discussions embodied in this article we begin with an overview of the PDT process, starting with a brief historical perspective followed by detailed discussions of specific applications of imaging in PDT. Each section starts with an overview of the specific topic and, where appropriate, ends with summary and future directions. The review closes with the authors’ perspective of the areas of future emphasis and promise. The basic premise of this review is that a combination of imaging and PDT will provide improved research and therapeutic strategies.

PDT is a photochemistry-based approach that uses a light-activatable chemical, termed a photosensitizer (PS), and light of an appropriate wavelength, to impart cytotoxicity via the generation of reactive molecular species (Figure 1a). In clinical settings, the PS is typically administered intravenously or topically, followed by illumination using a light delivery system suitable for the anatomical site being treated (Figure 1b). The time delay, often referred to as drug-light interval, between PS administration and the start of illumination with currently used PSs varies from 5 minutes to 24 hours or more depending on the specific PS and the target disease. Strictly speaking, this should be referred to as the PS-light interval, as at the concentrations typically used the PS is not a drug, but the drug-light interval terminology seems to be used fairly frequently. Typically, the useful range of wavelengths for therapeutic activation of the PS is 600 to 800 nm, to avoid interference by endogenous chromophores within the body, and yet maintain the energetics necessary for the generation of cytotoxic species (as discussed below) such as singlet oxygen (1O2). However, it is important to note that photosensitizers can also serve as fluorescence imaging agents for which activation with light in the 400nm range is often used and has been extremely useful in diagnostic imaging applications as described extensively in Section 2 of this review. The obvious limitation of short wavelength excitation is the lack of tissue penetration so that the volumes that are probed under these conditions are relatively shallow.






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Figure 1


(A) A schematic representation of PDT where PS is a photoactivatable multifunctional agent, which, upon light activation can serve as both an imaging agent and a therapeutic agent. (B) A schematic representation of the sequence of administration, localization and light activation of the PS for PDT or fluorescence imaging. Typically the PS is delivered systemically and allowed to circulate for an appropriate time interval (the “drug-light interval”), during which the PS accumulates preferentially in the target lesion(s) prior to light activation. In the idealized depiction here the PS is accumulation is shown to be entirely in the target tissue, however, even if this is not the case, light delivery confers a second layer of selectivity so that the cytotoxic effect will be generated only in regions where both drug and light are present. Upon localization of the PS, light activation will result in fluorescence emission which can be implemented for imaging applications, as well as generation cytotoxic species for therapy. In the former case light activation is achieved with a low fluence rate to generate fluorescence emission with little or no cytotoxic effect, while in the latter case a high fluence rate is used to generate a sufficient concentration of cytotoxic species to achieve biological effects.

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Imaging and photodynamic therapy: mechanisms, monitoring, and optimization.

BODIPY dyes tend to be highly fluorescent, but their emissions can be attenuated by adding substituents with appropriate oxidation potentials. Substituents like these have electrons to feed into photoexcited BODIPYs, quenching their fluorescence, thereby generating relatively long-lived triplet states. Singlet oxygen is formed when these triplet states interact with 3O2. In tissues, this causes cell damage in regions that are illuminated, and this is the basis of photodynamic therapy (PDT). The PDT agents that are currently approved for clinical use do not feature BODIPYs, but there are many reasons to believe that this situation will change. This review summarizes the attributes of BODIPY dyes for PDT, and in some related areas.

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BODIPY dyes in photodynamic therapy.

A positive temperature coefficient is the term which has been used to indicate that an increase in solubility occurs as the temperature is raised, whereas a negative coefficient indicates a decrease in solubility with rise in temperature.

Effect of Temperature

Human tumor cells of the lines WiDr (adenocarcinoma of the rectosigmoid colon), NHIK 3025 (carcinoma of the cervix), and V79 Chinese hamster fibroblasts were treated with 5-aminolevulinic acid (ALA) and ALA esterified to C1-C3 and C6-C8 chained aliphatic alcohols (ALA-esters). In the human cell lines, esterification of ALA with the long-chain (C6-C8) alcohols was found to reduce 30-150-fold the amount of ALA needed to reach the same level of protoporphyrin IX (PpIX) accumulation as with non-esterified ALA. The long-chained ALA-esters were less efficient in stimulating PpIX formation in V79 cells, i.e., the same amount of PpIX was formed by a 1-2.6-fold lower concentration of long-chained ALA-esters than with ALA. Short-chained ALA-esters (C1-C3) induced 5 to 10 times lower PpIX accumulation than ALA in all of the cell lines. High-performance liquid chromatography and fluorescence microscopic studies indicated that esterification of ALA has neither impact on the fluorescing porphyrin species formed nor impact on their intracellular localization. The PpIX formed from ALA-esters and ALA was found to be equally efficient in sensitizing cells to photoinactivation. The present results indicate that esterified ALAs are new and promising drugs for use in photochemotherapy of cancer.

https://cancerres.aacrjournals.org/content/canres/57/8/1481.full.pdf

Use of 5-aminolevulinic acid esters to improve photodynamic therapy on cells in culture.

Many medical applications, including photodynamic therapy for cancer (PDT), involve the use of lasers. However, the coherence of laser light is not necessary for PDT, and attempts have been made to construct non-coherent light sources for PDT, which are relatively inexpensive, stable and easy to operate, require simple maintenance but differ fundamentally from the lasers in their output characteristics. In the present work we compared two clinically used lamps, CureLight1, which is a broadband source (560–740 nm) based on a filtered halogen lamp, and CureLight2, which is a narrowband source based on light-emitting diodes (LEDs), with respect to several parameters of crucial significance for PDT efficiency in vivo: (a) depth of action in tissues, (b) heating effects, (c) pain generation, (d) photodegradation of PpIX in solution, in cells and in mouse skin and (e) photo-inactivation of cells in vitro. We conclude that CureLight2 (LED), relative to CureLight1 (halogen) has deeper PDT action in tissue, similar efficiency for bleaching PpIX in mouse skin, better efficiency for bleaching PpIX in cells and solutions and good efficiency for inactivating cells in vitro. CureLight2 gives less heating of the tissue and less pain in unsensitised human skin. All these differences are related to difference in the spectra of the lamps. Thus, PDT light sources with emissions that are visually similar have significantly different photobiological properties.

Effectiveness of different light sources for 5-aminolevulinic acid photodynamic therapy

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study.

By using a chemical extraction assay and confocal laser scanning fluorescence microscopy, the kinetic patterns of uptake, elimination, and localization of meso -tetra(hydroxyphenyl)porphine (m-THPP) and its corresponding chlorin (m-THPC) in tumors and various normal tissues of female C3D2/F1 mice bearing CaD2 mammary carcinoma were studied after an i.p. injection of either 5 mg/kg body weight of m-THPP or 1 mg/kg body weight of m-THPC. Moreover, the histological and ultrastructural alterations of the tumors were evaluated after photodynamic therapy (PDT) with m-THPP or m-THPC. The PDT efficacy with m-THPP and m-THPC was also compared. Both m-THPP and m-THPC had a similar kinetic pattern of distribution in the tumors and most normal tissues examined. The concentrations of the dyes in the tissues peaked at 24–48 h after injection. The peak values of the uptake of m-THPP by the tissues were found to decrease in the following order: spleen > urinary tract > kidney > liver > lung > tumor > heart > skin > muscle > brain. However, higher concentrations of m-THPC were taken up by the tumors than by most of the normal tissues studied except for the liver, urinary tract, and skin. m-THPP was mainly localized in the stroma of the tumors, whereas m-THPC was distributed in both vascular interstitium and neoplastic cells of the tumors. Morphological studies showed that PDT with m-THPP resulted in destructive changes in the microvasculature of the tumors, whereas m-THPC-based PDT destroyed both vascular walls and tumor cells of the tumors. The m-THPP-PDT of the tumors was much less efficient than m-THPC-PDT of the tumors, although the dose of m-THPP used was five times higher than that of m-THPC. m-THPP and m-THPC have different efficiency of sensitizing tumors to photodestruction, although they are similar with respect to hydrophobicity. This is likely due to the differences in their intratumoral localization patterns and in their absorption spectra.

https://cancerres.aacrjournals.org/content/canres/55/12/2620.full.pdf

Uptake, Localization, and Photodynamic Effect of meso-Tetra(hydroxyphenyl)porphine and Its Corresponding Chlorin in Normal and Tumor Tissues of Mice Bearing Mammary Carcinoma

The kinetics of protoporphyrin IX (PpIX) production in normal tissues and WiDr tumors of mice were studied after topical application of 5-aminolevulinic acid (ALA) and its methyl ester and hexyl ester. ALA and ALA esters were applied on a spot of 1.0 cm diameter on normal skin and on skin overlaying tumors. PpIX production was studied by fluorescence measurements. ALA induced PpIX not only on the spot of application but also on remote skin areas. This was not found for the ALA esters. They produced PpIX only on the spot of application. Thus, ALA, but neither its esters nor PpIX, is passing into the circulation. The time needed for ALA to enter the circulation through normal skin was about 5 hr. Even when looking normal, the skin overlaying tumors was more permeable to ALA than normal skin. Thus, when applied on the tumor, ALA induced PpIX on remote skin areas without any lag phase. Mainly, PpIX was found in all tissues although small amounts of a porphyrin with an excitation peak at about 400 nm, supposedly uroporphyrin and/or coproporphyrin, were found, notably in remote skin areas. An altered stratum corneum of the skin overlaying tumors probably contributes to the tumorselectivity, although in the present tumor system less PpIX was found in tumors than in muscles. This is probably related to biochemical and physiological conditions in this particular tumor, since i.p. injection of ALA also leads to less PpIX formation in the tumor than in skin/muscle tissue. Nevertheless, it seems evident that ALA can diffuse more easily from the skin surface and down to the vasculature in the tumor than in the normal tissue and that this leads to a higher concentration of PpIX in the tumor than would have been found if the physiological factors relevant for drug diffusion were the same for tumors as for skin/muscles. © 2001 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/1097-0215%28200102%299999%3A9999%3C%3A%3AAID-IJC1154%3E3.0.CO%3B2-K

Li Wei Ma

Papers

Use of 5-aminolevulinic acid esters to improve photodynamic therapy on cells in culture.

Effectiveness of different light sources for 5-aminolevulinic acid photodynamic therapy

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study.

Uptake, Localization, and Photodynamic Effect of meso-Tetra(hydroxyphenyl)porphine and Its Corresponding Chlorin in Normal and Tumor Tissues of Mice Bearing Mammary Carcinoma

On the pharmacokinetics of topically applied 5-aminolevulinic acid and two of its esters