Volume 5, Issue 2 - Topic Issue: From optical bioimaging to clinical biophotonics / Issue Editors: Mikhail Yu. Kirillin, Natalia M. Shakhova and Ilya V. Turchin

Background and objective: Pancreatic cancer has long been a leading cause of cancer death. Few patients are suitable for surgery and for those who are not, the response to treatment is generally poor. No more than about 10% survive for more than a year. Recent research has focused on focal treatment for local disease control. This review covers the development of one of the most promising options, photodynamic therapy (PDT). Methods: This review covers pre-clinical and clinical studies. Laboratory work was designed to understand the effect of PDT on the normal pancreas and surrounding tissues and on transplanted cancers in the hamster pancreas to ensure safety prior to clinical application. Essentially all clinical studies have been undertaken in University College Hospital, London. Phase-I studies used the photosensitisers mTHPC and verteporfin in patients with localised but inoperable cancers. Results: Laboratory results showed that normal pancreas, bile duct, liver, stomach and major blood vessels could tolerate PDT without any unacceptable effects on the structure and function of these organs. Necrosis that healed safely was documented in transplanted cancers. The clinical trials showed that focal necrosis could be produced in inoperable cancers with acceptable levels of complications, but considerable refinements of treatment delivery and monitoring are required before the technique will be ready for assessment in controlled clinical trials. Conclusions: PDT is showing promise for the minimally invasive treatment of localised pancreatic cancers, but it is still at an early stage of development. Much more work will be necessary to optimise techniques for applying PDT to these cancers and for combining it with other therapeutic options such as chemotherapy.

Background and objective: Photodynamic therapy (PDT) has been successfully used in clinical practice for decades; however, clinical outcome data are not always consistent resulting in a great necessity for real-time monitoring to predict the therapy outcome. Study design and methods: In a retrospective clinical study, 402 patients with non-melanoma skin malignancies were enrolled who underwent PDT treatment and fluorescence real-time imaging. The photosensitizer used was a chlorine e6 derivative (Fotoditazin ® ); the tumors were irradiated with a 662 nm continuous wave diode laser with fiber delivery system and total fluence of up to 300 J/cm 2 . The fluorescence imaging was performed using a commercially available system with a camera and bandpass filter in the range of 710–800 nm. Fluorescence contrast ( FC ) of the tumor (the ratio of the average fluorescence intensities in the tumor and the surrounding tissues) and its change during the PDT treatment (photobleaching, dFC ) was measured. Then the correlation between the clinical outcome (tumor response and recurrence rate) and measured fluorescence parameters was evaluated. The follow-up period was 6–53 months (median, 28 months). Results: FC or dFC below their median values independently correspond to a significant increase in tumor recurrence rate ( p <0.05), and slight increase of partial or no tumor response cases. Tumor response is better correlated with the value of FC , and not correlated with the photobleaching. Conclusion: Baseline FC and its change after PDT treatment may serve as a predictor of recurrence. This finding is a step towards individualized PDT cancer treatment.

Objective: A flow cytometric approach is proposed to assess the hydrogen peroxide (H 2 O 2 ) level under chemotherapy action separately in viable and apoptotic tumor cells. Materials and methods: For studying the involvement of H 2 O 2 in the process of cell death, the genetically encoded fluorescent sensor HyPer2, apoptosis marker PE Annexin V and vital dye 7-AAD were employed. The approach was used for testing the capacity of two cytotoxic drugs, cisplatin and bleomycin, to change the intracellular H 2 O 2 concentration, depending on the stage of cell death. Results: An increase in HyPer2 fluorescence has been revealed in cells undergoing apoptosis under cisplatin action. This finding indicates that accumulation of H 2 O 2 accompanies the cisplatin-induced apoptotic reaction. HyPer2 response was also revealed in negative to PE Annexin V viable cells which can be explained either by participation of H 2 O 2 in the earliest stages of apoptosis or in a cell response to a non-fatal injury. Under bleomycin action, neither an apoptotic reaction nor changes of fluorescence intensity HyPer2 were detected, allowing one to assume that H 2 O 2 is not involved in the reaction of tumor cells to bleomycin. Conclusion: The proposed approach can be used for studying the mechanisms of cell death under action of any types of antitumor drugs.

Background and objectives: Intensive research in the area of photodynamic therapy (PDT) has been made in recent years revealing it as a promising method for the treatment of tumors and inactivation of pathogenic microorganisms. However, for a broader application of this therapy one major challenge, namely a significant improvement of the targeted drug delivery and uptake, still remains. A possible solution of the selectivity problem could be the application of specifically functionalized photosensitizers, in particular phthalocyanine dyes. Materials and methods: Water-soluble Zn(II) phthalocyanines (ZnPcs) with four galactose moieties on non-peripheral and peripheral positions and a non-substituted Zn(II) phthalocyanine were studied for in vitro antitumor activity on three breast cancer cell lines (MCF-7, MDA-MB-231 and HBL-100). The influence of the exposure to ultraviolet (UV) (365 nm) and red (635 nm) light in non-therapeutic doses on the cellular uptake, binding and subcellular localization of three photosensitizers was investigated by confocal laser scanning microscopy. In addition, phototoxicity studies with the tested phthalocyanines on the non-tumorigenic mouse embryo cell line Balb c/3T3 (clone 31) were carried out. Results: The results indicate that the pre-treatment, namely exposure to UV or red light, influences the localization properties of the used dyes. The positions of galactose units to the ZnPc ring also influenced the uptake, localization and the photodynamic response of breast cancer cells. The results show that the galactose substitution, together with exposure to UV or red light in non-therapeutic doses, are important factors for the photodynamic effect. Conclusion: Experimental PDT with galactose-substituted ZnPcs accompanied by UV and red light pre-irradiation leads to a higher photodynamic effect towards breast tumor cells. Thus, the investigated galactopyranosyl-substituted phthalocyanines could be used as a part of the design of intelligent, stimuli-responsive nanosystems for medical applications.

Background and objective: Endoscopic laser lithotripsy is the preferred technique for minimally invasive destruction of ureteral and kidney stones, and is mostly performed by pulsed holmium:yttrium-aluminum-garnet (Ho:YAG) laser irradiation. The absorbed laser energy heats the water creating a vapor bubble which collapses after the laser pulse, thus producing a shock wave. Part of the laser energy strikes the stone through the vapor bubble and induces thermomechanical material removal. Aim of the present study was to visualize the behavior and the dynamics of the cavitation bubble using a specially developed ultra-short-time illumination system and then to determine important characteristics related to clinically used laser and application parameters for a more detailed investigation in the future. Materials and methods: In accordance with Toepler’s Schlieren technique, in the ultra-short-time-illumination set-up the cavitation bubble which had been induced by Ho:YAG laser irradiation at the fiber end, was illuminated by two Q-switched lasers and the process was imaged in high contrast on a video camera. Cavitation bubbles were induced using different pulse energies (500 mJ/pulse and 2000 mJ/pulse) and fiber core diameters (230 μm and 600 μm) and the bubble dynamics were recorded at different times relative to the Ho:YAG laser pulse. The time-dependent development of the bubble formation was determined from the recordings by measuring the bubble diameter in horizontal and vertical directions, together with the volume and localization of the center of the bubble collapse. Results: The results show that the bubble dynamics can be visualized and studied with both high contrast and high temporal resolution. The bubble volume increases with pulse energy and with fiber diameter. The bubble shape is almost round when a larger fiber core diameter is used, and elliptical when using a fiber of smaller core diameter. Moreover, the center of the resulting bubble is slightly further away from the fiber end and the center of the bubble collapse for a smaller fiber core diameter. Conclusion: The experimental set-up developed gives a better understanding of the bubble dynamics. The experiments indicate that the distance between fiber tip and target surface, as well as the laser parameters used have considerable impact on the cavitation bubble dynamics. Both the bubble dynamics and their influence on the stone fragmentation process require further investigation.

Objective: The results of a feasibility study of the application of PEG-300 and fructose as two independent optical clearing agents for the reduction of light scattering in biological tissues are presented. Materials and methods: An OCT system operating at 1300 nm was used to study optical clearing effects. In in-vitro experiments in mice (n=2) an increase of the imaging depth was observed after intravenous injection of PEG-300 alone and in combination with intradermal injection of fructose. The optical clearing effect was also studied for the first time in two mice in vivo using intravenous injection of PEG-300 or solution of hemoglobin. Results: The intradermal injection of fructose in combination with the intravenous injection of PEG-300 led to a rapid optical clearing effect. In the experiments on mice in vivo the injection of PEG-300 or hemoglobin solution into the tail vein of the living mice allowed for a rapid enhancement of the vein wall and the surrounding tissue image contrast. Conclusion: The experiments on mice have clearly demonstrated that intradermal and intravenous injections of optical clearing agents enhanced light transport through the skin and blood vessels.

Abstract: Optical coherence tomography (OCT) is a modern technique for imaging of internal structures of biotissue of up to several millimeters in depth with a resolution of several micrometers. However, various external conditions can distort the diagnostic capabilities of an OCT image. Mechanical compression and temperature regime are the two conditions which mostly affect the diagnostic OCT images obtained with a contact probe. It is shown here that the application of compression to human skin induces a decrease in contrast of the stratum corneum-epidermis junction and an increase in contrast of the epidermis-dermis junction. With regard to these junctions, a preliminary change of biotissue temperature induces additional changes in the contrast, with opposing effects in case of heating and cooling.