Synthesis, characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate.

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by (1)H NMR, UV-vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2mWcm(-2)) of red light (643nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC50 values. The obtained IC50 values for 5 were 1.4µM at 15min irradiation time and 0.7µM when the time of irradiation was 30min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.

Optical characterization and reverse engineering based on multiangle spectroscopy.

We perform characterization of thin films and reverse engineering of multilayer coatings on the basis of multiangle spectral photometric data provided by a new advanced spectrophotometer accessory. Experimental samples of single thin films and multilayer coatings are produced by magnetron sputtering and electron-beam evaporation. Reflectance and transmittance data at two polarization states are measured at incidence angles from 7 to 40 deg. We demonstrate that multiangle reflectance and transmittance data provide reliable characterization and reverse-engineering results.

Comparison of two techniques for reliable characterization of thin metal-dielectric films.

In the present study we determine the optical parameters of thin metal-dielectric films using two different characterization techniques based on nonparametric and multiple oscillator models. We consider four series of thin metal-dielectric films produced under various deposition conditions with different optical properties. We compare characterization results obtained by nonparametric and multiple oscillator techniques and demonstrate that the results are consistent. The consistency of the results proves their reliability.

General approach to reliable characterization of thin metal films.

Optical constants of thin metal films are strongly dependent on deposition conditions, growth mode, and thickness. We propose a universal characterization approach that allows reliable determination of thin metal film optical constants as functions of wavelength and thickness. We apply this approach to determination of refractive index dispersion of silver island films embedded between silica layers.

Gradient silver nanoparticle layers in absorbing coatings--experimental study.

Metal island films show a characteristic absorption peak related to the surface plasmon resonance of free electrons. This kind of film can be used in absorbing coatings, together with dielectric layers. Such absorbing multilayer coatings, with and without the gradient of the silver mass thickness in metal island films throughout the coating, have been deposited by electron beam evaporation. It is shown experimentally that coatings with a gradient in the mass thickness of silver nanoparticles have higher absorption than equivalent nongradient coatings with the same total mass thickness of silver nanoparticles.

Use of gold island films in design of reflectors with high luminosity.

We describe the optical properties of gold island films embedded between SiO2 and/or TiO2 layers. Plasmonic properties of gold films have been characterized using spectrometry and variable angle spectroscopic ellipsometry for various combinations of the embedding media. The obtained refractive indices of embedded gold island films have been used in the design of several types of multilayer reflectors.

On the dielectric function tuning of random metal-dielectric nanocomposites for metamaterial applications.

The potential of random metal-dielectric nanocomposites as constituent elements of metamaterial structures is explored. Classical effective medium theories indicate that these composites can provide a tunable negative dielectric function with small absorption losses. However, the tuning potential of real random composites is significantly lower than the one predicted by classical theories, due to the underestimation of the spectral range where topological resonances take place. This result suggests that a random mixture consisting of a metal matrix with embedded isolated dielectric inclusions is a promising design guideline for the fabrication of tunable composites for metamaterial purposes.

Fractionated illumination improves the outcome in the treatment of precancerous lesions with photodynamic therapy.

Photodynamic therapy (PDT) is a noninvasive therapy for non-melanoma skin cancer. The aim of this study was comparison of efficacy between fractioned versus single dose illumination in photodynamic therapy (PDT) of actinic keratosis (AK) and Bowen's disease (BD). Fifty-one patients (36 AK and 15 BD) were treated with PDT They were randomly arranged in two treatment groups. Group one included 26 patients (20 AK and 6 BD) that, after five hours of incubation with 20% 5-ALA, were treated with a single illumination of 100 Jcm(-2) at fluence rate of 30 mWcm(-2). Group two included 25 patients (16 AK and 9 BD) that, after 16 hours of incubation with 20% 5-ALA, were treated with two light fractions (50 plus 50 Jcm(-2)) at same fluence rate with dark interval of two hours between fractions. Twenty-four weeks later, a treated area was incubated for four hours again with 5-ALA in order to detect occult areas of abnormal skin with possible remaining tumor tissue. In case of fluorescence, histological examination was performed. In the group one, fluorescence at the end of the session was absent in 19 (73%) or very weak in 7 (27%). Residual tumor was found in 15 (75%) AK and in 4 (66.6%) BD. In the group two, fluorescence at the end of second session was more intense; in one patient (4%) was absent, very weak in 5 (20%) and weak in 19 (76%) of patients. In this group histology revealed remaining tumor tissue in only 2 (12.5%) AK and 2 (22.2%) BD. Among the patients in the first group, the remaining tumor tissue was significantly bigger (p=0.005). The treatment response with clearing of tumor tissue was significantly higher in fractionated illumination than in a single dose illumination group. Fractionated illumination scheme with 16 hours of incubation separated by two hours dark interval significantly improves the therapeutic outcome in tumor eradication.

Visualization of basal cell carcinoma by fluorescence diagnosis and independent component analysis.

Photodynamic detection (PDD) of skin tumours is based on the visualization of a fluorophores, with the ability to accumulate in tumour tissue, by the use of fluorescence imaging. Of particular importance is the application of δ-5-aminolaevulinic acid (ALA) that, through the process of biosynthesis causes formation of the protoporphyrin IX (PpIX). The PpIX has the ability of selective fluorescence after basal cell carcinoma (BCC) has been treated with ALA. Higher concentration of PpIX in tumour tissue compared to surrounding normal skin is the basis for PDD. Our contribution in this preliminary study is application of the independent component analysis (ICA) to extract the BCC spatial map, by processing fluorescent RGB image acquired under excitation with 405nm light. Comparative performance analysis with other two widely used image processing methods: ratio imaging and optimal threshold based imaging, reveals that ICA produces BCC spatial map that is most consistent in term of diagnostic quality by both visual assessment and calculation of the BCC demarcation line. We believe this represents a solid basis for the design of a compact and low-cost multi-spectral fluorescence imaging system, capable for real time calculation of the skin tumour demarcation.