Volume 4, Issue 1 - Topic Issue: Photodynamic diagnosis and therapy / Issue Editors: Stephen G. Bown and Herbert Stepp

5-Aminolevulinic acid (ALA)-induced photodynamic therapy (ALA-PDT) has achieved remarkable research accomplishments over the past 30 years, yet its application in medical oncology still awaits clear recognition as a valid alternative therapeutic modality. It is well documented that topical ALA-PDT enables the treatment of multiple skin lesions simultaneously, and provides excellent cosmetic results with no acquired multi-drug resistance (MDR). Furthermore, upon disease recurrence the treatment can be repeated resulting in the same therapeutic efficacy. Additionally, in oncological surgery, ALA fluorescence-guided resection is a practical and simple method for visualizing intra-operative brain and urological tumors with millimeter accuracy. The urgent challenge is to direct future research of ALA-phototherapy and fluorescence diagnosis to the maturation of their medical status in oncology. Therefore, the future objectives are to amplify critical evidence-based results of ALA-PDT safety and efficacy and to validate its unique advantages over other technologies. Strong statistical PDT documentation and the positive predictive values of protoporphyrin IX (PpIX)-guided surgery will persuade the medical community to implement ALA-based therapeutics into standard clinical and surgical oncology practice. Research must address the phenomenon that no MDR develops as a consequence of PDT, since MDR is the major stumbling block in oncological therapeutics. A feasible goal should be to improve ALA administration protocols based on recent knowledge that preactivation of the enzyme porphobilinogen deaminase enhances PpIX accumulation in cancer cells and photodestruction. Moreover the recent introduction of multifunctional ALA prodrugs that maximize photosensitizer biosynthesis, targeting multiple sub-cellular targets, may increase PDT anti-cancer efficacy in additional disease settings. In conclusion, well-documented clinical results, new ALA delivery protocols, and novel multifunctional ALA prodrugs may advance ALA-PDT to becoming a front-line cancer therapy.

The prodrug 5-aminolevulinic acid (ALA) and its ester derivatives have been used in photodynamic therapy (PDT) in dermatology worldwide. In China, ALA-PDT was first used to treat urethral condylomata acuminata and non-melanoma skin cancers in 1997. A powder formulation of ALA hydrochloride was approved by the Chinese Food and Drug Administration for the treatment of condylomata acuminata in 2007. Large successful experience of treating condylomatas was accumulated compared with Western countries. Meanwhile, numerous clinical studies as well as off-label use of ALA-PDT have been carried out in China. To reflect the progress of ALA-PDT in China, several major Chinese and English databases were searched and published data were reviewed in this article.

Background: Protoporphyrin IX (PpIX)-induced photodynamic therapy (PDT) is being utilised as a topical method of localised ablation of certain non-melanoma skin cancers and precancers. Standardised protocols have been implemented to good effect when the disease remains superficial but improvement is required to treat thicker or acrally located conditions. Concurrent administration of an iron chelator during PpIX-PDT has been demonstrated to increase cellular accumulation of PpIX by reducing its bioconversion to haem (an iron dependent process) thus increasing cell kill on subsequent irradiation. Iron however, can also play a role in reactive oxygen species (ROS) generation and limiting its availability via chemical chelation could theoretically reduce the efficacy of PpIX-PDT, so that a response less than that maximally feasible is produced. Materials and methods: The effects of iron availability and chelation on PpIX-PDT have therefore been investigated via fluorescence quantification of PpIX accumulation, single-cell gel electrophoresis (comet assay) measurement of ROS-induced DNA damage and trypan blue exclusion assessment of cell viability. Cultured human cells were utilised and incubated in standardised iron conditions with the PpIX precursor’s aminolaevulinic acid (ALA) or its methyl ester (MAL) in the presence or absence of either of the iron chelating agents desferrioxamine (DFO) or hydroxypyridinone (CP94), or alternatively iron sulphate as a source of iron. Results: ALA or MAL incubation was found to significantly increase cellular PpIX accumulation pre-irradiation as anticipated and this observation correlated with both significantly increased DNA damage and reduced cellular viability following irradiation. Co-incubation with either of the iron chelators investigated (DFO or CP94) significantly increased pre-irradiation PpIX accumulation as well as DNA damage and cell death on irradiation indicating the positive effect of iron chelation on the effectiveness of PpIX-induced PDT. The opposite effects were observed however, when the cells were co-incubated with iron sulphate, with significant reductions in pre-irradiation PpIX accumulation (ALA only) and DNA damage (ALA and MAL) being recorded indicating the negative effects excessive iron can have on PpIX-PDT effectiveness. Some dark toxicity produced by iron sulphate administration in non-irradiated control groups was also observed. Conclusion: Iron chelation and availability have therefore been observed to positively and adversely affect the PpIX-PDT process respectively and it is concluded that the effects of increased PpIX accumulation pre-irradiation produced via iron chelation outweigh any limitations reduced iron availability may have on the ability of iron to catalyse ROS generation/cascades following PpIX-induced PDT. Further investigation of iron chelation within dermatological applications where enhanced PpIX-PDT treatment effects would be beneficial is therefore warranted.

Objective: Dosimetry of singlet oxygen ( 1 O 2 ) is of particular interest because it is the major cytotoxic agent causing biological effects of type-II photosensitizers during photodynamic therapy (PDT). An in-vivo model was developed to determine the singlet oxygen threshold dose, [ 1 O 2 ] rx,sh , for PDT. 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 [ 1 O 2 ] 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. In order to discuss the possible influence of vascular vs. apoptotic cell killing mechanisms on the singlet oxygen threshold dose, the [ 1 O 2 ] rx,sh values for BPD with 3 h and 15 min drug-light intervals, with the latter being known to have a dominantly vascular effect, were compared. Results: The mean values (standard deviation) of the in-vivo [ 1 O 2 ] rx,sh are approximately 0.56 (0.26) and 0.72 (0.21) m m (or 3.6×10 7 and 4.6×10 7 singlet oxygen per cell to reduce the cell survival to 1/ e ) for Photofrin ® and BPD 3 h , respectively, assuming that the fraction of generated singlet oxygen that interacts with the cell is 1. The [ 1 O 2 ] rx,sh value for BPD 15 min (0.12) was substantially lower than that for a DLI of 3 h. 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 [ 1 O 2 ] rx,sh . For mTHPC-PDT, the [ 1 O 2 ] rx,sh value derived from in-vivo mouse study was reported to be 0.4 m m . In comparison, the singlet oxygen required per cell was reported to be 9×10 8 per cell per 1/ e fractional kill in an in-vitro mTHPC-PDT study on a rat prostate cancer cell line (MLL cells) and was reported to be 7.9 m m for a multicell in-vitro EMT6/Ro spheroid model for mTHPC-PDT. Conclusions: The experimental results of [ 1 O 2 ] rx,sh 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 the knowledge that factors other than singlet oxygen-mediated tumor cell killing can contribute to PDT damage in-vivo .

Near-infrared (NIR) dyes are used as fluorescence markers in small animal imaging and in diffuse optical tomography. In these applications it is important to know whether the dyes bind to proteins or to other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. Unfortunately, neither the optical beam paths nor the detectors of commonly used in confocal and multiphoton laser scanning microscopes (LSMs) directly allow for excitation and detection of NIR fluorescence. This paper presents three ways of adapting existing LSMs with time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) systems for NIR FLIM: 1) confocal systems with wideband beamsplitters and diode laser excitation, 2) confocal systems with wideband beamsplitters and one-photon excitation by titanium-sapphire lasers, and 3) two-photon systems with optical parametric oscillator (OPO) excitation and non-descanned detection. A number of NIR dyes are tested in biological tissue. All of them show clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information about the tissue composition and on local biochemical parameters.

Background: Fluorescence diffuse tomography (FDT) is the most accurate technique for the imaging of labeled tumors in the small animal body. However, the procedure for reconstruction of the spatial distribution of the fluorophore requires a high signal-to-noise ratio due to the ill-condition of the inverse problem. Therefore, the FDT technique is ineffective for imaging tumors of small size or with dim fluorophores because of the low intensity of their fluorescence compared with the high level of tissue autofluorescence. In these cases, the size and position of a marked tumor in the animal body can be estimated from two-dimensional fluorescence images obtained using trans- or epi-illumination techniques. Material and methods: A versatile system for small animal fluorescence imaging which combines planar epi- and trans-illumination geometries of the light source and of the fluorescence receiver was created and tested. For epi-illumination imaging, light-emitting diode sources were used to provide homogeneous and stable illumination of the experimental animal, in combination with a cooled CCD camera which covers the entire illuminated area. For trans-illumination imaging, mechanical raster-scanning devices modulated at a low frequency were used for the laser source, together with a cooled photomultiplier tube, which provided outstanding sensitivity. Results: Monitoring the orthotopic tumor growth in animal bodies has demonstrated the efficacy of trans-illumination imaging in comparison with the epi-illumination technique. The results obtained also showed that the effective use of the trans-illumination technique requires Born normalization of the fluorescence signal and the exclusion of lateral illumination by surrounding the animal with additional light absorption material using light-absorption pads on both sides of the body.

Background and objective: Enamel irradiation with an erbium,chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser has been reported to cause micromorphological alterations within resin-enamel interfaces and subsurface enamel. The present study was conducted to evaluate the effects of different output power/pulse repetition rate parameter combinations of Er,Cr:YSGG laser on the micromorphology of resin-enamel interfaces and subsurface enamel. Materials and methods: Thirty-five bovine incisors were used in the present study. Er,Cr:YSGG laser was used on enamel with different output power/pulse repetition rate parameter combinations as follows: 6 W/20 Hz, 6 W/35 Hz, 6 W/50 Hz, 3 W/20 Hz, 3 W/35 Hz, 3 W/50 Hz. Following adhesive and composite procedures, specimens were sectioned and either acid-etched with 37% phosphoric acid for 1 min or immersed in 55% nitric acid for 12 h. Then the resin-enamel interfaces were investigated by scanning electron microscopy (SEM). Results: Micromorphological findings by SEM revealed that when enamel surfaces were irradiated with all 6-W groups and 3 W/20 Hz-combinations, large resin structures indicating subsurface microcracks were generally evident within interfaces and subsurface enamel. However, when irradiated with 3 W/35 Hz and 3 W/50 Hz-combinations, the observed resin-enamel interfaces were similar to those of the acid-etched only group. Conclusion: Deploying Er,Cr:YSGG laser with lower power and high pulse repetition rate combinations may reduce damage to resin-enamel interfaces and subsurface enamel. However, assessment of the relationship between these findings and bond strength requires further study.